|
1
|
Genetic Background Influences Severity of Colonic Aganglionosis and Response to GDNF Enemas in the Holstein Mouse Model of Hirschsprung Disease. Int J Mol Sci 2021; 22:ijms222313140. [PMID: 34884944 PMCID: PMC8658428 DOI: 10.3390/ijms222313140] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2021] [Revised: 11/30/2021] [Accepted: 12/03/2021] [Indexed: 12/13/2022] Open
Abstract
Hirschsprung disease is a congenital malformation where ganglia of the neural crest-derived enteric nervous system are missing over varying lengths of the distal gastrointestinal tract. This complex genetic condition involves both rare and common variants in dozens of genes, many of which have been functionally validated in animal models. Modifier loci present in the genetic background are also believed to influence disease penetrance and severity, but this has not been frequently tested in animal models. Here, we addressed this question using Holstein mice in which aganglionosis is due to excessive deposition of collagen VI around the developing enteric nervous system, thereby allowing us to model trisomy 21-associated Hirschsprung disease. We also asked whether the genetic background might influence the response of Holstein mice to GDNF enemas, which we recently showed to have regenerative properties for the missing enteric nervous system. Compared to Holstein mice in their original FVB/N genetic background, Holstein mice maintained in a C57BL/6N background were found to have a less severe enteric nervous system defect and to be more responsive to GDNF enemas. This change of genetic background had a positive impact on the enteric nervous system only, leaving the neural crest-related pigmentation phenotype of Holstein mice unaffected. Taken together with other similar studies, these results are thus consistent with the notion that the enteric nervous system is more sensitive to genetic background changes than other neural crest derivatives.
Collapse
|
|
2
|
Seruggia D, Josa S, Fernández A, Montoliu L. The structure and function of the mouse tyrosinase locus. Pigment Cell Melanoma Res 2020; 34:212-221. [DOI: 10.1111/pcmr.12942] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2020] [Revised: 10/06/2020] [Accepted: 10/14/2020] [Indexed: 12/23/2022]
Affiliation(s)
- Davide Seruggia
- Department of Molecular and Cellular Biology National Centre for Biotechnology (CNB‐CSIC) Madrid Madrid Spain
- CIBERER‐ISCIII Madrid Spain
- Division of Hematology/Oncology Boston Children's HospitalHarvard Medical School Boston MA USA
| | - Santiago Josa
- Department of Molecular and Cellular Biology National Centre for Biotechnology (CNB‐CSIC) Madrid Madrid Spain
- CIBERER‐ISCIII Madrid Spain
| | - Almudena Fernández
- Department of Molecular and Cellular Biology National Centre for Biotechnology (CNB‐CSIC) Madrid Madrid Spain
- CIBERER‐ISCIII Madrid Spain
| | - Lluis Montoliu
- Department of Molecular and Cellular Biology National Centre for Biotechnology (CNB‐CSIC) Madrid Madrid Spain
- CIBERER‐ISCIII Madrid Spain
| |
Collapse
|
|
3
|
Li H, Shao F, Qian B, Sun Y, Huang Z, Ding Z, Dong L, Chen J, Zhang J, Zang Y. Upregulation of HER2 in tubular epithelial cell drives fibroblast activation and renal fibrosis. Kidney Int 2019; 96:674-688. [DOI: 10.1016/j.kint.2019.04.012] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2018] [Revised: 03/12/2019] [Accepted: 04/05/2019] [Indexed: 12/20/2022]
|
|
4
|
Pilon N. Pigmentation-based insertional mutagenesis is a simple and potent screening approach for identifying neurocristopathy-associated genes in mice. Rare Dis 2016; 4:e1156287. [PMID: 27141416 PMCID: PMC4838316 DOI: 10.1080/21675511.2016.1156287] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2015] [Accepted: 02/12/2016] [Indexed: 01/28/2023] Open
Abstract
Neurocristopathies form a specific group of rare genetic diseases in which a defect in neural crest cell development is causal. Because of the large number of neural crest cell derivatives, distinct structures/cell types (isolated or in combination) are affected in each neurocristopathy. The most important issues in this research field is that the underlying genetic cause and associated pathogenic mechanism of most cases of neurocristopathy are poorly understood. This article describes how a relatively simple insertional mutagenesis approach in the mouse has proved useful for identifying new candidate genes and pathogenic mechanisms for diverse neurocristopathies.
Collapse
Affiliation(s)
- Nicolas Pilon
- Molecular Genetics of Development Laboratory, Department of Biological Sciences and BioMed Research Center, Faculty of Sciences, University of Quebec at Montreal (UQAM), Montreal, Quebec, Canada; UQAM Research Chair on Rare Genetic Diseases, Montreal, Canada
| |
Collapse
|
|
5
|
Ohtsuka M, Miura H, Mochida K, Hirose M, Hasegawa A, Ogura A, Mizutani R, Kimura M, Isotani A, Ikawa M, Sato M, Gurumurthy CB. One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT). BMC Genomics 2015; 16:274. [PMID: 25887549 PMCID: PMC4404087 DOI: 10.1186/s12864-015-1432-5] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2015] [Accepted: 03/04/2015] [Indexed: 01/10/2023] Open
Abstract
BACKGROUND The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently. RESULTS Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice. CONCLUSIONS The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.
Collapse
Affiliation(s)
- Masato Ohtsuka
- Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
| | - Hiromi Miura
- Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
| | - Keiji Mochida
- RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan.
| | - Michiko Hirose
- RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan.
| | - Ayumi Hasegawa
- RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan.
| | - Atsuo Ogura
- RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan. .,Graduate School of Life and Environmental Science, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki, 305-8572, Japan.
| | - Ryuta Mizutani
- Graduate School of Engineering, Tokai University, Kitakaname, Hiratsuka, Kanagawa, 259-1292, Japan.
| | - Minoru Kimura
- Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
| | - Ayako Isotani
- Immunology Frontier Research Center, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
| | - Masahito Ikawa
- Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
| | - Masahiro Sato
- Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima, 890-8544, Japan.
| | - Channabasavaiah B Gurumurthy
- Mouse Genome Engineering Core Facility, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, 68198, USA.
| |
Collapse
|
|
6
|
Weinstein MM, Tompson SW, Chen Y, Lee B, Cohn DH. Mice expressing mutant Trpv4 recapitulate the human TRPV4 disorders. J Bone Miner Res 2014; 29:1815-1822. [PMID: 24644033 PMCID: PMC4108531 DOI: 10.1002/jbmr.2220] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/09/2013] [Revised: 02/21/2014] [Accepted: 03/06/2014] [Indexed: 11/08/2022]
Abstract
Activating mutations in transient receptor potential vanilloid family member 4 (Trpv4) are known to cause a spectrum of skeletal dysplasias ranging from autosomal dominant brachyolmia to lethal metatropic dysplasia. To develop an animal model of these disorders, we created transgenic mice expressing either wild-type or mutant TRPV4. Mice transgenic for wild-type Trpv4 showed no morphological changes at embryonic day 16.5 but did have a delay in bone mineralization. Overexpression of a mutant TRPV4 caused a lethal skeletal dysplasia that phenocopied many abnormalities associated with metatropic dysplasia in humans, including dumbbell-shaped long bones, a small ribcage, abnormalities in the autopod, and abnormal ossification in the vertebrae. The difference in phenotype between embryos transgenic for wild-type or mutant Trpv4 demonstrates that an increased amount of wild-type protein can be tolerated and that an activating mutation of this protein is required to produce a skeletal dysplasia phenotype.
Collapse
Affiliation(s)
- Michael M Weinstein
- Department of Molecular, Cell, and Developmental Biology, Orthopaedic Hospital Research Center, University of California, Los Angeles, CA 90095.,Department of Orthopaedic Surgery and Orthopaedic Hospital Research Center, University of California, Los Angeles, CA 90095
| | - Stuart W Tompson
- Department of Molecular, Cell, and Developmental Biology, Orthopaedic Hospital Research Center, University of California, Los Angeles, CA 90095
| | - Yuqing Chen
- Howard Hughes Medical Institute and Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030
| | - Brendan Lee
- Howard Hughes Medical Institute and Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030
| | - Daniel H Cohn
- Department of Molecular, Cell, and Developmental Biology, Orthopaedic Hospital Research Center, University of California, Los Angeles, CA 90095.,Department of Orthopaedic Surgery and Orthopaedic Hospital Research Center, University of California, Los Angeles, CA 90095
| |
Collapse
|
|
7
|
Insertional mutagenesis by a hybrid piggyBac and sleeping beauty transposon in the rat. Genetics 2012; 192:1235-48. [PMID: 23023007 DOI: 10.1534/genetics.112.140855] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat.
Collapse
|
|
8
|
Cornett JC, Landrette SF, Xu T. Characterization of fluorescent eye markers for mammalian transgenic studies. PLoS One 2011; 6:e29486. [PMID: 22216292 PMCID: PMC3247282 DOI: 10.1371/journal.pone.0029486] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2011] [Accepted: 11/29/2011] [Indexed: 11/18/2022] Open
Abstract
Genotyping mice by DNA based methods is both laborious and costly. As an alternative, we systematically examined fluorescent proteins expressed in the lens as transgenic markers for mice. A set of eye markers has been selected such that double and triple transgenic animals can be visually identified and that fluorescence intensity in the eyes can be used to distinguish heterozygous from homozygous mice. Taken together, these eye markers dramatically reduce the time and cost of genotyping transgenics and empower analysis of genetic interaction.
Collapse
Affiliation(s)
- Jonathan C. Cornett
- Department of Genetics, Boyer Center for Molecular Medicine, Yale University School of Medicine, Howard Hughes Medical Institute, New Haven, Connecticut, United States of America
| | - Sean F. Landrette
- Department of Genetics, Boyer Center for Molecular Medicine, Yale University School of Medicine, Howard Hughes Medical Institute, New Haven, Connecticut, United States of America
| | - Tian Xu
- Department of Genetics, Boyer Center for Molecular Medicine, Yale University School of Medicine, Howard Hughes Medical Institute, New Haven, Connecticut, United States of America
- Institute of Developmental Biology and Molecular Medicine, Fudan-Yale Center for Biomedical Research, School of Life Sciences, Fudan University, Shanghai, China
- * E-mail:
| |
Collapse
|
|
9
|
Wang B, Harrison W, Overbeek PA, Zheng H. Transposon mutagenesis with coat color genotyping identifies an essential role for Skor2 in sonic hedgehog signaling and cerebellum development. Development 2011; 138:4487-97. [PMID: 21937600 PMCID: PMC3177318 DOI: 10.1242/dev.067264] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/10/2011] [Indexed: 01/15/2023]
Abstract
Correct development of the cerebellum requires coordinated sonic hedgehog (Shh) signaling from Purkinje to granule cells. How Shh expression is regulated in Purkinje cells is poorly understood. Using a novel tyrosinase minigene-tagged Sleeping Beauty transposon-mediated mutagenesis, which allows for coat color-based genotyping, we created mice in which the Ski/Sno family transcriptional co-repressor 2 (Skor2) gene is deleted. Loss of Skor2 leads to defective Purkinje cell development, a severe reduction of granule cell proliferation and a malformed cerebellum. Skor2 is specifically expressed in Purkinje cells in the brain, where it is required for proper expression of Shh. Skor2 overexpression suppresses BMP signaling in an HDAC-dependent manner and stimulates Shh promoter activity, suggesting that Skor2 represses BMP signaling to activate Shh expression. Our study identifies an essential function for Skor2 as a novel transcriptional regulator in Purkinje cells that acts upstream of Shh during cerebellum development.
Collapse
Affiliation(s)
- Baiping Wang
- Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA
| | - Wilbur Harrison
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA
| | - Paul A. Overbeek
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
| | - Hui Zheng
- Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
| |
Collapse
|
|
10
|
Hamilton SM, Spencer CM, Harrison WR, Yuva-Paylor LA, Graham DF, Daza RA, Hevner RF, Overbeek PA, Paylor R. Multiple autism-like behaviors in a novel transgenic mouse model. Behav Brain Res 2011; 218:29-41. [PMID: 21093492 PMCID: PMC3022332 DOI: 10.1016/j.bbr.2010.11.026] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2010] [Revised: 11/05/2010] [Accepted: 11/09/2010] [Indexed: 11/21/2022]
Abstract
Autism spectrum disorder (ASD) diagnoses are behaviorally based with no defined universal biomarkers, occur at a 1:110 ratio in the population, and predominantly affect males compared to females at approximately a 4:1 ratio. One approach to investigate and identify causes of ASD is to use organisms that display abnormal behavioral responses that model ASD-related impairments. This study describes a novel transgenic mouse, MALTT, which was generated using a forward genetics approach. It was determined that the transgene integrated within a non-coding region on the X chromosome. The MALTT line exhibited a complete repertoire of ASD-like behavioral deficits in all three domains required for an ASD diagnosis: reciprocal social interaction, communication, and repetitive or inflexible behaviors. Specifically, MALTT male mice showed deficits in social interaction and interest, abnormalities in pup and juvenile ultrasonic vocalization communications, and exhibited a repetitive stereotypy. Abnormalities were also observed in the domain of sensory function, a secondary phenotype prevalently associated with ASD. Mapping and expression studies suggested that the Fam46 gene family may be linked to the observed ASD-related behaviors. The MALTT line provides a unique genetic model for examining the underlying biological mechanisms involved in ASD-related behaviors.
Collapse
Affiliation(s)
- Shannon M. Hamilton
- Department of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Corinne M. Spencer
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Wilbur R. Harrison
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Lisa A. Yuva-Paylor
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Deanna F. Graham
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Ray A.M. Daza
- Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, WA 98101, USA
| | - Robert F. Hevner
- Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, WA 98101, USA
- Department of Neurological Surgery, University of Washington School of Medicine, Seattle, WA 98104, USA
| | - Paul A. Overbeek
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Richard Paylor
- Department of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA
| |
Collapse
|
|
11
|
Yu EM, Ye X, Wang HY, Bai JJ, Xia SL, Lao HH, Jian Q. Isolation of Tanichthys albonubes beta actin gene and production of transgenic Tanichthys albonubes. FISH PHYSIOLOGY AND BIOCHEMISTRY 2010; 36:173-180. [PMID: 20467859 DOI: 10.1007/s10695-008-9238-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/27/2008] [Accepted: 05/27/2008] [Indexed: 05/29/2023]
Abstract
A beta actin cDNA of Tanichthys albonubes was isolated through the RT-PCR and RACE approach. The cDNA was 1,787-bp in length, including a 1,128-bp CDS, a 95-bp 5'UTR and a 564-bp 3'UTR. Genomic DNA containing the transcription region and 5'-flanking region was cloned based on the beta actin cDNA by Genome walker. A 3,000-bp beta actin gene promoter was then produced by PCR according to the sequences of the 5'-flanking region and the first intron. This promoter consisted of a 1,800-bp 5'-flanking region, and a 1,200-bp 5'-UTR. 3 transcription elements, CAAT box, CArG motif and TATA box were found in the 5'-flanking region. This promoter was inserted into the vector pDsRed2-1 and microinjected into fertilized eggs of Tanichthys albonubes to prove its transcription activity. The beta actin promoter and GH CDS of Tanichthys albonubes were then fused to construct an expression vector pTLA-GH. GH-transgenic Tanichthys albonubes was obtained by microinjection of the pTLA-GH into the fertilized eggs. Fast-growth individuals were observed in the transgenic group and the body weight of the largest individual was 2.1-fold that of the maximum in its non-transgenic siblings in 100 dph. In addition, a co-injection strategy was employed with pTLA-DsRed and pTLA-GH vector and proven to enhance the efficiency of GH-transgenic fish detection.
Collapse
Affiliation(s)
- Er-meng Yu
- College of Aqua-life Science and Technology, Shanghai Ocean University, Shanghai 200090, China
| | | | | | | | | | | | | |
Collapse
|
|
12
|
Li W, Puertollano R, Bonifacino JS, Overbeek PA, Everett ET. Disruption of the murine Ap2β1 gene causes nonsyndromic cleft palate. Cleft Palate Craniofac J 2010; 47:566-73. [PMID: 20500056 DOI: 10.1597/09-145] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Development of the secondary palate in mammals is a complex process that can be easily perturbed, leading to the common and distressing birth defect cleft palate. Animal models are particularly useful tools for dissecting underlying genetic components of cleft palate. We describe a new cleft palate model resulting from a transgene insertion mutation. Transgene insertional mutagenesis disrupts the genomic organization and expression of the Ap2β1 gene located on chromosome 11. This gene encodes the β2-adaptin subunit of the heterotetrameric adaptor protein 2 complex involved in clathrin-dependent endocytosis. Homozygous cleft palate mutant mice express no Ap2β1 messenger RNA or β2-adaptin protein and die during the perinatal period. Heterozygous mice are phenotypically normal despite expressing diminished β2-adaptin messenger RNA and protein compared with wildtype. Remarkably, the paralogous β1-adaptin subunit of the adaptor protein 1 complex partially substitutes for the missing β2-adaptin in embryonic fibroblasts from homozygous mutant mice, resulting in assembly of reduced levels of an adaptor protein 2 complex bearing β1-adaptin. This variant adaptor protein 2 complex is, therefore, apparently capable of maintaining viability of the homozygous mutant embryos until birth but insufficient to support palatogenesis. Nonsyndromic cleft palate in an animal model is associated with disruption of the Ap2β1 gene.
Collapse
Affiliation(s)
- Wei Li
- Department of Oral Facial Development, Indiana University School of Dentistry, Indianapolis, Indiana, USA
| | | | | | | | | |
Collapse
|
|
13
|
Bouma GJ, Hudson QJ, Washburn LL, Eicher EM. New candidate genes identified for controlling mouse gonadal sex determination and the early stages of granulosa and Sertoli cell differentiation. Biol Reprod 2009; 82:380-9. [PMID: 19864314 DOI: 10.1095/biolreprod.109.079822] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022] Open
Abstract
Mammalian gonadal sex-determining (GSD) genes are expressed in a unique population of somatic cells that differentiate into granulosa cells in XX gonads or Sertoli cells in XY gonads. The ability to efficiently isolate these somatic support cells (SSCs) during the earliest stages of gonad development would facilitate identifying 1) new candidate GSD genes that may be involved in cases of unexplained abnormal gonad development and 2) genes involved in the earliest stages of granulosa and Sertoli cell differentiation. We report the development of a unique mouse carrying two transgenes that allow XX and XY mice to be distinguished as early as Embryonic Day 11.5 (E11.5) and allow SSCs to be isolated from undifferentiated (E11.5) and early differentiated (E12.5) fetal gonads. The Mouse Genome 430v2.0 GeneChip (Affymetrix) was used to identify transcripts exhibiting a sexual dimorphic expression pattern in XX and XY isolated SSCs. The analysis revealed previously unidentified sexually dimorphic transcripts, including low-level expressed genes such as Sry, a gene not identified in other microarray studies. Multigene real-time PCR analysis of 57 genes verified that 53 were expressed in fetal gonads in a sexually dimorphic pattern, and whole-mount in situ hybridization analysis verified 4930563E18Rik, Pld1, and Sprr2d are expressed in XX gonads, and Fbln2, Ppargc1a, and Scrn1 are expressed in XY gonads. Taken together, the data provide a comprehensive resource for the spatial-temporal expression pattern of genes that are part of the genetic network underlying the early stages of mammalian fetal gonadal development, including the development of granulosa and Sertoli cells.
Collapse
|
|
14
|
Grueninger F, Bohrmann B, Czech C, Ballard TM, Frey JR, Weidensteiner C, von Kienlin M, Ozmen L. Phosphorylation of Tau at S422 is enhanced by Abeta in TauPS2APP triple transgenic mice. Neurobiol Dis 2009; 37:294-306. [PMID: 19781645 DOI: 10.1016/j.nbd.2009.09.004] [Citation(s) in RCA: 75] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2009] [Revised: 09/01/2009] [Accepted: 09/10/2009] [Indexed: 10/20/2022] Open
Abstract
Amyloid beta peptides and microtubule-associated protein Tau are misfolded and form aggregates in brains of Alzheimer's disease patients. To examine their specific roles in the pathogenesis of Alzheimer's disease and their relevance in neurodegenerative processes, we have created TauPS2APP triple transgenic mice that express human mutated Amyloid Precursor Protein, presenilin 2 and Tau. We present a cross-sectional analysis of these mice at 4, 8, 12 and 16 months of age. By comparing with single transgenic Tau mice, we demonstrate that accumulation of Abeta in TauPS2APP triple transgenic mice impacts on Tau pathology by increasing the phosphorylation of Tau at serine 422, as determined by a novel immunodetection method that is able to reliably measure phospho-Tau species in transgenic mouse brains. The TauPS2APP triple transgenic mouse model will be very useful for studying the effect of new therapeutic paradigms on amyloid deposition and downstream neurofibrillary tangle development.
Collapse
Affiliation(s)
- Fiona Grueninger
- F. Hoffmann-La-Roche Ltd, Pharmaceutical Research Neuroscience, Basel, Switzerland
| | | | | | | | | | | | | | | |
Collapse
|
|
15
|
Abstract
Chromosomal rearrangements, such as deletions, duplications, inversions and translocations, occur frequently in humans and can be disease-associated or phenotypically neutral. To understand the genetic consequences of such genomic changes, these mutations need to be modelled in experimentally tractable systems. The mouse is an excellent organism for this analysis because of its biological and genetic similarity to humans, the ease with which its genome can be manipulated and the similarity of observed affects. Through chromosome engineering, defined rearrangements can be introduced into the mouse genome. The resulting mouse models are leading to a better understanding of the molecular and cellular basis of dosage alterations in human disease phenotypes, in turn opening new diagnostic and therapeutic opportunities.
Collapse
Affiliation(s)
- Louise van der Weyden
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, UK
| | | | | |
Collapse
|
|
16
|
Trimboli AJ, Fukino K, de Bruin A, Wei G, Shen L, Tanner SM, Creasap N, Rosol TJ, Robinson ML, Eng C, Ostrowski MC, Leone G. Direct evidence for epithelial-mesenchymal transitions in breast cancer. Cancer Res 2008; 68:937-45. [PMID: 18245497 DOI: 10.1158/0008-5472.can-07-2148] [Citation(s) in RCA: 280] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
We developed stromal- and epithelial-specific cre-transgenic mice to directly visualize epithelial-mesenchymal transition (EMT) during cancer progression in vivo. Using three different oncogene-driven mouse mammary tumor models and cell-fate mapping strategies, we show in vivo evidence for the existence of EMT in breast cancer and show that myc can specifically elicit this process. Hierarchical cluster analysis of genome-wide loss of heterozygosity reveals that the incidence of EMT in invasive human breast carcinomas is rare, but when it occurs it is associated with the amplification of MYC. These data provide the first direct evidence for EMT in breast cancer and suggest that its development is favored by myc-initiated events.
Collapse
Affiliation(s)
- Anthony J Trimboli
- Department of Molecular Genetics, College of Medicine, The Ohio State University, Columbus, OH 43210, USA
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
17
|
Jang CW, Behringer RR. Transposon-mediated transgenesis in rats. Cold Spring Harb Protoc 2007; 2007:pdb.prot4866. [PMID: 21356954 DOI: 10.1101/pdb.prot4866] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/30/2023]
Abstract
INTRODUCTIONThis protocol describes a method for the production of transgenic rats by coinjecting circular PiggyBac transposon-containing plasmid DNA with transposase-encoding mRNA. After fertilized eggs are collected from females, pronuclei are microinjected with the DNA and mRNA and then transferred immediately into the oviducts of pseudopregnant foster females. Using this procedure, we have obtained transformation frequencies of 33%-100% (average of ~80%). Traditional methods for generating transgenic rats (i.e., microinjection of linear DNA) yield 3%-41% transgenic founders. Thus, transposon-mediated transformation of fertilized rat eggs is an efficient alternative method to generate transgenic rats.
Collapse
Affiliation(s)
- Chuan-Wei Jang
- Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA
| | | |
Collapse
|
|
18
|
Xiao D, Yue Y, Deng XY, Huang B, Guo ZM, Ma Y, Lin YL, Hong X, Tang H, Xu K, Chen XG. Rescue of the albino phenotype by introducing a functional tyrosinase minigene into Kunming albino mice. World J Gastroenterol 2007; 13:244-9. [PMID: 17226903 PMCID: PMC4065952 DOI: 10.3748/wjg.v13.i2.244] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To use the tyrosinase minigene as a visual marker to perform microinjection training and improve the techniques related with transgene to greatly elevate the efficiency of gene transfer.
METHODS: A mouse tyrosinase minigene, i.e., TyBS, in which the 2.25-kb authentic genomic 5’ non-coding flanking sequence of mouse tyrosinase was fused to a mouse tyrosinase cDNA, was introduced into the fertilized eggs of outbred Kunming albino mice.
RESULTS: Of the 11 animals that developed from the injected eggs, two mice (P1 and #8) exhibited pigmented hair (P1) and eyes (P1 and #8), as confirmed by PCR analysis for the tyrosinase minigene integrated into the genome. When founder P1 was bred to Kunming male mouse, six progeny out of 11 offspring inherited the transgene and the pigmented-eye phenotype.
CONCLUSION: Taken together, these results suggest that this minigene encodes the active tyrosinase protein and that its 5’ flanking region contains the sequences regulating the expression of mouse tyrosinase gene as expected. We have rescued the albino phenotype by introduction and expression of a functional tyrosinase minigene in the Kunming albino mouse and the transgene can be passed to subsequent generation. These findings also indicate that TyBS can be a useful visual marker gene in the co-transgenic experiments.
Collapse
Affiliation(s)
- Dong Xiao
- Center of Experimental Animals, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
19
|
Guo KK, Ren J. Cardiac overexpression of alcohol dehydrogenase (ADH) alleviates aging-associated cardiomyocyte contractile dysfunction: role of intracellular Ca2+ cycling proteins. Aging Cell 2006; 5:259-65. [PMID: 16842498 DOI: 10.1111/j.1474-9726.2006.00215.x] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Aging is a complex biological process with contributions from a wide variety of genes including insulin-like growth factor I and alcohol dehydrogenase (ADH), which decline with advanced age. The goal of this study was to examine if ADH enzyme plays any role in cardiac aging. Ventricular myocytes were isolated from young (2-3 months old) or aged (26-28 months old) male FVB wild-type and cardiac-specific ADH (class I, isozyme type 1) transgenic mice. Mechanical properties were measured using an IonOptix system. Aged FVB myocytes displayed significantly reduced ADH activity compared with young ones, which was restored by the ADH transgene. Compared with young cardiomyocytes, aged FVB myocytes exhibited prolonged relengthening duration and a steaper decline in peak shortening amplitude in response to elevated electrical stimuli. Although ADH transgene itself did not alter mechanical properties in young mice, it rescued aging-associated diastolic dysfunction without affecting dampened contractile response to high stimulus frequency. Immunoblot analysis revealed reduced sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) and Na(+)-Ca(2+) exchanger (NCX) levels in conjunction with enhanced phospholamban expression in aged FVB hearts. ADH transgene prevented aging-induced reduction in SERCA2a and NCX without affecting up-regulated phospholamban. Our data suggest that aging is associated with a reduced ADH enzymatic activity and diastolic dysfunction, which may be corrected with cardiac overexpression of the ADH enzyme. Alteration in cardiac Ca(2+) cycling proteins including SERCA2a and NCX may play a role in both pathogenesis of cardiac aging and the beneficial effect of ADH enzyme.
Collapse
Affiliation(s)
- Kelly K Guo
- Division of Pharmaceutical Sciences & Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, WY 82071, USA
| | | |
Collapse
|
|
20
|
Shen X, Zheng S, Metreveli NS, Epstein PN. Protection of cardiac mitochondria by overexpression of MnSOD reduces diabetic cardiomyopathy. Diabetes 2006; 55:798-805. [PMID: 16505246 DOI: 10.2337/diabetes.55.03.06.db05-1039] [Citation(s) in RCA: 287] [Impact Index Per Article: 15.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
We previously reported damage and elevated biogenesis in cardiac mitochondria of a type 1 diabetic mouse model and proposed that mitochondria are one of the major targets of oxidative stress. In this study, we targeted overexpression of the mitochondrial antioxidant protein manganese superoxide dismutase (MnSOD) to the heart to protect cardiac mitochondria from oxidative damage. Transgenic hearts had a 10- to 20-fold increase in superoxide dismutase (SOD) activity, and the transgenic SOD was located in mitochondria. The transgene caused a twofold increase in cardiac catalase activity. MnSOD transgenic mice demonstrated normal cardiac morphology, contractility, and mitochondria, and their cardiomyocytes were protected from exogenous oxidants. Crossing MnSOD transgenic mice with our type 1 model tested the benefit of eliminating mitochondrial reactive oxygen species. Overexpression of MnSOD improved respiration and normalized mass in diabetic mitochondria. MnSOD also protected the morphology of diabetic hearts and completely normalized contractility in diabetic cardiomyocytes. These results showed that elevating MnSOD provided extensive protection to diabetic mitochondria and provided overall protection to the diabetic heart.
Collapse
Affiliation(s)
- Xia Shen
- Department of Pathology, University of Washington, Seattle, USA
| | | | | | | |
Collapse
|
|
21
|
Christensen R, Alhonen L, Wahlfors J, Jakobsen M, Jensen TG. Characterization of transgenic mice with the expression of phenylalanine hydroxylase and GTP cyclohydrolase I in the skin. Exp Dermatol 2005; 14:535-42. [PMID: 15946242 DOI: 10.1111/j.0906-6705.2005.00326.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
Phenylketonuria (PKU) is a metabolic disease causing increased levels of phenylalanine in blood and body fluids. Circulating phenylalanine is normally cleared by phenylalanine hydroxylase (PAH) expressed in the liver. The aim of this study is to exploit the skin as a 'metabolic sink' removing phenylalanine from the blood. We have previously showed that the overexpression of PAH and GTP cyclohydrolase I (GTP-CH), the rate-limiting enzyme in the synthesis of the cofactor for PAH, leads to high levels of phenylalanine clearance in primary human keratinocytes. In this study, we have investigated the 'metabolic sink' strategy in an in vivo model by developing three lines of transgenic mice expressing PAH and GTP-CH in various layers of the skin. The promoters used were keratin 14 (K14), involucrin (INV) and a truncated variant of Keratin 1 (K1). The mice were crossbred to a mouse model of human PKU, the PAH(enu2) mouse, in order to obtain mice that do not express PAH in the liver and the kidney. Transgenic mice containing the INV and K14 promoters expressed PAH and GTP-CH in the epidermis. However, the K1 promoter did not lead to detectable gene expression. Analysis of the mice showed that no phenotypic effect was observed in mice expressing PAH and GTP-CH from the INV promoter. However, low level of phenylalanine clearance was observed in mice expressing PAH and GTP-CH from the K14 promoter, suggesting that the skin can be genetically engineered to function as a 'metabolic sink'.
Collapse
Affiliation(s)
- Rikke Christensen
- Department of Human Genetics, University of Aarhus, Aarhus, Denmark.
| | | | | | | | | |
Collapse
|
|
22
|
Zhang X, Dong F, Li Q, Borgerding AJ, Klein AL, Ren J. Cardiac overexpression of catalase antagonizes ADH-associated contractile depression and stress signaling after acute ethanol exposure in murine myocytes. J Appl Physiol (1985) 2005; 99:2246-54. [PMID: 16109828 DOI: 10.1152/japplphysiol.00750.2005] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Alcohol dehydrogenase (ADH), which oxidizes ethanol into acetaldehyde, exacerbates ethanol-induced cardiac depression, although the mechanism of action remains unclear. This study was designed to examine the impact of antioxidant catalase (CAT) on cardiac contractile response to ethanol and activation of stress signaling. ADH-CAT double transgenic mice were generated by crossing CAT and ADH lines. Mechanical, intracellular Ca(2+) properties and reactive oxygen species generation were measured in ventricular myocytes. ADH-CAT, ADH, CAT and wild-type FVB myocytes exhibited similar mechanical and intracellular Ca(2+) properties. ADH or ADH-CAT myocytes had higher acetaldehyde-producing ability. Ethanol (80-640 mg/dl) suppressed FVB cell shortening and intracellular Ca(2+) transients with maximal inhibitions of 43.5 and 45.2%, respectively. Ethanol-induced depression on cell shortening and intracellular Ca(2+) was augmented in ADH group with maximal inhibitions of 66.8 and 69.6%, respectively. Interestingly, myocytes from CAT-ADH mice displayed normal ethanol response with maximal inhibitions of 46.0 and 47.2% for cell shortening and intracellular Ca(2+), respectively. CAT transgene lessened ethanol-induced inhibition on cell shortening (maximal inhibition of 30.3%) but not intracellular Ca(2+). ADH amplified ethanol-induced reactive oxygen species generation, which was nullified by the CAT transgene. Western blot analysis showed that ethanol reduced ERK phosphorylation and enhanced JNK phosphorylation without affecting p38 phosphorylation. The ethanol-induced changes in phosphorylation of ERK and JNK were amplified by ADH. CAT transgene itself did not affect ethanol-induced response in ERK and JNK phosphorylation, but it cancelled ADH-induced effects. These data suggest that antioxidant CAT may effectively antagonize ADH-induced enhanced cardiac depression in response to ethanol.
Collapse
Affiliation(s)
- Xiaochun Zhang
- Division of Pharmaceutical Sciences & Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, WY 82071, USA
| | | | | | | | | | | |
Collapse
|
|
23
|
Otsuki A, Tahimic CGT, Tomimatsu N, Katoh M, Chen DJ, Kurimasa A, Oshimura M. Construction of a novel expression system on a human artificial chromosome. Biochem Biophys Res Commun 2005; 329:1018-25. [PMID: 15752757 DOI: 10.1016/j.bbrc.2005.02.079] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2005] [Indexed: 11/21/2022]
Abstract
Efficient regulation of transgene would greatly facilitate the analysis of gene function in biological systems for basic research and clinical applications. The tetracycline-regulatable system (TRS) has proven to be a promising tool for such purposes. Despite their widespread application, a number of challenges are still associated with the use of TRS, including clonal variability in the regulation and copy number. We have recently constructed a novel human artificial chromosome (HAC) called 21DeltaqHAC. By housing a TRS-based DNA-PKcs expression cassette in this HAC, we were able to circumvent the problems associated with conventional TRS-based vectors. We achieved tight control of DNA-PKcs expression and rescued the radiosensitive phenotype of DNA-PKcs-deficient CHO cells. The combined use of HAC and the TRS serves as a model for controllable and fixed copy number expression vectors. Our study also demonstrates the suitability of the HAC to accommodate multi-subunit constructs such as that of the TRS.
Collapse
Affiliation(s)
- Akihiro Otsuki
- Department of Genetic Medicine and Regenerative Therapeutics, Graduate School of Medical Sciences, Tottori University, 86 Nishicho, Yonago, Tottori 683-8503, Japan
| | | | | | | | | | | | | |
Collapse
|
|
24
|
Wolf SE, Woodside KJ. Transgenic and gene knock-out techniques and burn research. J Surg Res 2005; 123:328-39. [PMID: 15680397 DOI: 10.1016/j.jss.2004.06.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2004] [Indexed: 02/03/2023]
Abstract
The development of transgenic technology has given researchers a powerful tool to examine biological effects, and the response to injury is no exception. Techniques such as pronuclear injection, targeted homologous recombination, and Cre/loxP gene excision are being used to construct animals with specific genetic designs; these are exploited to learn the role of genes in the response to severe burn. We review the construction of transgenic animals, pitfalls and benefits of this relatively new technique, and how this technique has been used in burn research.
Collapse
Affiliation(s)
- Steven E Wolf
- Department of Surgery, University of Texas Health Science Center--San Antonio, San Antonio, Texas, USA.
| | | |
Collapse
|
|
25
|
Giménez E, Lavado A, Giraldo P, Cozar P, Jeffery G, Montoliu L. A Transgenic Mouse Model with Inducible Tyrosinase Gene Expression Using the Tetracycline (Tet-on) System Allows Regulated Rescue of Abnormal Chiasmatic Projections Found in Albinism. ACTA ACUST UNITED AC 2004; 17:363-70. [PMID: 15250938 DOI: 10.1111/j.1600-0749.2004.00158.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET-ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.
Collapse
Affiliation(s)
- Estela Giménez
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, 28049 Madrid, Spain
| | | | | | | | | | | |
Collapse
|
|
26
|
Kuklin AI, Mynatt RL, Klebig ML, Kiefer LL, Wilkison WO, Woychik RP, Michaud EJ. Liver-specific expression of the agouti gene in transgenic mice promotes liver carcinogenesis in the absence of obesity and diabetes. Mol Cancer 2004; 3:17. [PMID: 15175105 PMCID: PMC443512 DOI: 10.1186/1476-4598-3-17] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2004] [Accepted: 06/02/2004] [Indexed: 11/25/2022] Open
Abstract
Background The agouti protein is a paracrine factor that is normally present in the skin of many species of mammals. Agouti regulates the switch between black and yellow hair pigmentation by signalling through the melanocortin 1 receptor (Mc1r) on melanocytes. Lethal yellow (Ay) and viable yellow (Avy) are dominant regulatory mutations in the mouse agouti gene that cause the wild-type protein to be produced at abnormally high levels throughout the body. Mice harboring these mutations exhibit a pleiotropic syndrome characterized by yellow coat color, obesity, hyperglycemia, hyperinsulinemia, and increased susceptibility to hyperplasia and carcinogenesis in numerous tissues, including the liver. The goal of this research was to determine if ectopic expression of the agouti gene in the liver alone is sufficient to recapitulate any aspect of this syndrome. For this purpose, we generated lines of transgenic mice expressing high levels of agouti in the liver under the regulatory control of the albumin promoter. Expression levels of the agouti transgene in the liver were quantified by Northern blot analysis. Functional agouti protein in the liver of transgenic mice was assayed by its ability to inhibit binding of the α-melanocyte stimulating hormone (αMSH) to the Mc1r. Body weight, plasma insulin and blood glucose levels were analyzed in control and transgenic mice. Control and transgenic male mice were given a single intraperitoneal injection (10 mg/kg) of the hepatocellular carcinogen, diethylnitrosamine (DEN), at 15 days of age. Mice were euthanized at 36 or 40 weeks after DEN injection and the number of tumors per liver and total liver weights were recorded. Results The albumin-agouti transgene was expressed at high levels in the livers of mice and produced a functional agouti protein. Albumin-agouti transgenic mice had normal body weights and normal levels of blood glucose and plasma insulin, but responded to chemical initiation of the liver with an increased number of liver tumors compared to non-transgenic control mice. Conclusions The data demonstrate that liver-specific expression of the agouti gene is not sufficient to induce obesity or diabetes, but, in the absence of these factors, agouti continues to promote hepatocellular carcinogenesis.
Collapse
Affiliation(s)
- Alexander I Kuklin
- Life Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831, USA
- Transgenomic, Inc., 12325 Emmet Street, Omaha, NE 68164, USA
| | - Randall L Mynatt
- Life Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831, USA
- Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808, USA
| | - Mitchell L Klebig
- Life Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831, USA
- Department of Biochemistry and Cellular & Molecular Biology, The University of Tennessee, Knoxville, TN 37996, USA
| | - Laura L Kiefer
- Glaxo Wellcome, 5 Moore Drive, Research Triangle Park, NC 27709, USA
- Paradigm Genetics, 108 Alexander Drive, Research Triangle Park, NC 27709, USA
| | - William O Wilkison
- Glaxo Wellcome, 5 Moore Drive, Research Triangle Park, NC 27709, USA
- GlaxoSmithKline, Inc., 5 Moore Drive, Research Triangle Park, NC 27709, USA
| | - Richard P Woychik
- Life Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831, USA
- The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA
| | - Edward J Michaud
- Life Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831, USA
| |
Collapse
|
|
27
|
Ye G, Metreveli NS, Ren J, Epstein PN. Metallothionein prevents diabetes-induced deficits in cardiomyocytes by inhibiting reactive oxygen species production. Diabetes 2003; 52:777-83. [PMID: 12606520 DOI: 10.2337/diabetes.52.3.777] [Citation(s) in RCA: 143] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Many individuals with diabetes experience impaired cardiac contractility that cannot be explained by hypertension and atherosclerosis. This cardiomyopathy may be due to either organ-based damage, such as fibrosis, or to direct damage to cardiomyocytes. Reactive oxygen species (ROS) have been proposed to contribute to such damage. To address these hypotheses, we examined contractility, Ca(2+) handling, and ROS levels in individual cardiomyocytes isolated from control hearts, diabetic OVE26 hearts, and diabetic hearts overexpressing antioxidant protein metallothionein (MT). Our data showed that diabetic myocytes exhibited significantly reduced peak shortening, prolonged duration of shortening/relengthening, and decreased maximal velocities of shortening/relengthening as well as slowed intracellular Ca(2+) decay compared with control myocytes. Overexpressing MT prevented these defects induced by diabetes. In addition, high glucose and angiotensin II promoted significantly increased generation of ROS in diabetic cardiomyocytes. Chronic overexpression of MT or acute in vitro treatment with the flavoprotein inhibitor diphenyleneiodonium or the angiotensin II type I receptor antagonist losartan eliminated excess ROS production in diabetic cardiomyocytes. These data show that diabetes induces damage at the level of individual myocyte. Damage can be attributed to ROS production, and diabetes increases ROS production via angiotensin II and flavoprotein enzyme-dependent pathways.
Collapse
Affiliation(s)
- Gang Ye
- Department of Pediatrics, University of Louisville School of Medicine, 570 S. Preston Street, Louisville, KY 40202, USA
| | | | | | | |
Collapse
|
|
28
|
Duan J, Esberg LB, Ye G, Borgerding AJ, Ren BH, Aberle NS, Epstein PN, Ren J. Influence of gender on ethanol-induced ventricular myocyte contractile depression in transgenic mice with cardiac overexpression of alcohol dehydrogenase. Comp Biochem Physiol A Mol Integr Physiol 2003; 134:607-14. [PMID: 12600669 DOI: 10.1016/s1095-6433(02)00347-1] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Acute ethanol exposure depresses ventricular contractility and contributes to alcoholic cardiomyopathy in both men and women chronically consuming ethanol. However, a gender-related difference in the severity of myopathy exists with female being more sensitive to ethanol-induced tissue damage. Acetaldehyde (ACA), the major oxidized product of ethanol, has been implicated to play a role in the pathogenesis and gender-related difference of alcoholic cardiomyopathy, possibly due to its direct cardiac effect and interaction with estrogen. This study was designed to compare the effects of cardiac overexpression of alcohol dehydrogenase (ADH), which converts ethanol into ACA, on the cardiac contractile response to ethanol in ventricular myocytes isolated from age-matched adult male and female transgenic (ADH) and wild-type (FVB) mice. Mechanical properties were measured with an IonOptix SoftEdge system. ACA production was assessed by gas chromatography. The ADH myocytes from both genders exhibited similar mechanical properties but a higher efficacy to produce ACA compared to FVB myocytes. Exposure to ethanol (80-640 mg/dl) for 60 min elicited concentration-dependent decrease of cell shortening in both FVB and ADH groups. The ethanol-induced depression on cell shortening was significantly augmented in female but not male ADH group. ADH transgene did not exacerbate the ethanol-induced inhibition of maximal velocity of shortening/relengthening in either gender. In addition, neither ethanol nor ADH transgene affect the duration of shortening and relengthening in male or female mice. These data suggest that females may be more sensitive to ACA-induced cardiac contractile depression than male, which may attribute to the gender-related difference of alcoholic cardiomyopathy.
Collapse
Affiliation(s)
- Jinhong Duan
- Division of Pharmaceutical Sciences, University of Wyoming College of Health Sciences, Laramie, WY 82071-3375, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
29
|
Aberle II NS, Ren J. Experimental Assessment of the Role of Acetaldehyde in Alcoholic Cardiomyopathy. Biol Proced Online 2003; 5:1-12. [PMID: 12734561 PMCID: PMC150386 DOI: 10.1251/bpo41] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2002] [Revised: 11/27/2002] [Accepted: 12/12/2002] [Indexed: 01/17/2023] Open
Abstract
Alcoholism is one of the major causes of non-ischemic heart damage. The myopathic state of the heart due to alcohol consumption, namely alcoholic cardiomyopathy, is manifested by cardiac hypertrophy, compromised ventricular contractility and cardiac output. Several mechanisms have been postulated for alcoholic cardiomyopathy including oxidative damage, accumulation of triglycerides, altered fatty acid extraction, decreased myofilament Ca(2+ )sensitivity, and impaired protein synthesis. Despite intensive efforts to unveil the mechanism and ultimate toxin responsible for alcohol-induced cardiac toxicity, neither has been clarified thus far. Primary candidates for the specific toxins are ethanol, its first and major metabolic product - acetaldehyde (ACA) and fatty acid ethyl esters. Evidence from our lab suggests that ACA directly impairs cardiac function and promotes lipid peroxidation resulting in oxidative damage. The ACA-induced cardiac contractile depression may be reconciled with inhibitors of Cytochrome P-450 oxidase, xanthine oxidase and lipid peroxidation Unfortunately, the common methods to investigate the toxicity of ACA have been hampered by the fact that direct intake of ACA is toxic and unsuitable for chronic study, which is unable to provide direct evidence of direct cardiac toxicity for ACA. In order to overcome this obstacle associated with the chemical properties of ACA, our laboratory has used the chronic ethanol feeding model in transgenic mice with cardiac over-expression of alcohol dehydrogenase (ADH) and an in vitro ventricular myocyte culture model. The combination of both in vivo and in vitro approaches allows us to evaluate the role of ACA in ethanol-induced cardiac toxicity and certain cellular signaling pathways leading to alcoholic cardiomyopathy.
Collapse
Affiliation(s)
- Nicholas S. Aberle II
- Department of Pharmacology, Physiology and Therapeutics, University of North Dakota School of Medicine and Health Sciences. Grand Forks, ND 58203. USA
| | - Jun Ren
- Division of Pharmaceutical Sciences, School of Pharmacy, University of Wyoming College of Health Sciences. Laramie, WY 82071-3375. USA. Phone: 307-766-6131 Fax: 307-766-2953
| |
Collapse
|
|
30
|
Abstract
In the postgenomic era the mouse will be central to the challenge of ascribing a function to the 40,000 or so genes that constitute our genome. In this review, we summarize some of the classic and modern approaches that have fueled the recent dramatic explosion in mouse genetics. Together with the sequencing of the mouse genome, these tools will have a profound effect on our ability to generate new and more accurate mouse models and thus provide a powerful insight into the function of human genes during the processes of both normal development and disease.
Collapse
|
|
31
|
Kubo J, Yamanouchi K, Naito K, Tojo H. Expression of the gene of interest fused to the EGFP-expressing gene in transgenic mice derived from selected transgenic embryos. THE JOURNAL OF EXPERIMENTAL ZOOLOGY 2002; 293:712-8. [PMID: 12410599 DOI: 10.1002/jez.10163] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
The present paper describes the expression of a target fusion gene, WAP/hGH fused to the EGFP-expressing gene in transgenic mice derived from the transfer of transgenic embryos selected because of their expression of enhanced green fluorescent protein (EGFP). The 6.7-kb fusion gene was microinjected as a single cassette gene construct into the pronuclei of mouse zygotes. The surviving embryos were cultured and were classified according to the EGFP expression patterns at the morula or blastocyst stage. After the transfer of embryos with uniform-expression or mosaic-expression of EGFP, transgenesis occurred in 85.7% to 86% or 44.1% to 44% of the pups, respectively. No transgenic pups were derived from EGFP negative embryos. In the transgenic females, EGFP was ubiquitously expressed under the control of the CAG promoter, and hGH was expressed under the control of the WAP promoter in an appropriate fashion: hGH was secreted into the milk of lactating transgenic females. The presence or absence of the expression of EGFP coincided with that of the hGH gene in the transgenic mice. The present cassette gene construct is a useful example for circumventing the routine analyses of DNA and RNA required for the generation and maintenance of transgenic lines.
Collapse
Affiliation(s)
- Jun Kubo
- Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
| | | | | | | |
Collapse
|
|
32
|
Giraldo P, Montoliu L. Artificial chromosome transgenesis in pigmentary research. PIGMENT CELL RESEARCH 2002; 15:258-64. [PMID: 12100491 DOI: 10.1034/j.1600-0749.2002.02030.x] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Pigmentary genes were among the first mammalian genes to be studied, mostly because of the obvious phenotypes associated with their mutations. In 1990, tyrosinase, encoding the limiting enzyme in the melanin synthesis pathway, was eventually assigned to the c (albino) locus by classical rescue experiments driven by functional constructs in transgenic mice. These pioneer reports triggered the study of the regulation of endogenous tyrosinase gene expression by combining different amounts of upstream regulatory and promoter regions and testing their function in vivo in transgenic animals. However, faithful and reproducible transgenic expression was not achieved until the entire tyrosinase expression domain was transferred to the germ-line of mice using artificial-chromosome-type transgenes. The use of these large tyrosinase transgenic constructs and the ease with which they could be manipulated in vitro enabled the discovery of previously unknown but fundamental regulatory regions, such as the tyrosinase locus control region (LCR), whose presence was required in order to guarantee position-independent and copy-number-dependent expression of tyrosinase transgenes, with an expression level, per copy, comparable to that of an endogenous wild-type allele. Subsequently, functional dissection of elements present within this LCR through the generation of new artificial-chromosome type tyrosinase transgenes has revealed the existence of different regulatory activities. The existence of some of these units had been suggested previously by standard-type transgenic analyses. In this review, we will discuss both independent approaches and conclude that optimal tyrosinase transgene expression requires the use of its complete expression domain.
Collapse
MESH Headings
- Animals
- Binding Sites/genetics
- Chromosomes, Artificial, Mammalian/genetics
- Chromosomes, Artificial, Mammalian/metabolism
- Gene Expression Regulation, Enzymologic/genetics
- Humans
- Melanocytes/enzymology
- Mice
- Mice, Transgenic/genetics
- Mice, Transgenic/metabolism
- Monophenol Monooxygenase/genetics
- Monophenol Monooxygenase/metabolism
- Pigments, Biological/biosynthesis
- Pigments, Biological/genetics
- Promoter Regions, Genetic/genetics
- Transgenes/genetics
Collapse
Affiliation(s)
- Patricia Giraldo
- Centro Nacional de Biotecnología (CNB-CSIC), Department of Molecular and Cellular Biology, Campus de Cantoblanco, Madrid, Spain
| | | |
Collapse
|
|
33
|
Hasegawa K, Nakatsuji N. Insulators prevent transcriptional interference between two promoters in a double gene construct for transgenesis. FEBS Lett 2002; 520:47-52. [PMID: 12044868 DOI: 10.1016/s0014-5793(02)02761-8] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
In transgenesis, the expression of two transgenes is often subject to mutual interference by each of the two expression cassettes when they are driven by different transcriptional regulatory elements in a single construct. To study this problem, we constructed vectors consisting of two expression units, one contains a strong ubiquitous promoter and the other contains a tissue-specific transcriptional element. The expression pattern of each transgene was examined in transfected cell lines and also in transgenic mice. In both cases, two expression units in a single construct were expressed in an independent manner and were controlled by their respective regulatory element only if we placed insulators at both ends of one expression unit. These results indicate that usage of insulators is a valuable tool for transfection of double gene constructs in transgenesis.
Collapse
Affiliation(s)
- Kouichi Hasegawa
- Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
| | | |
Collapse
|
|
34
|
Duan J, McFadden GE, Borgerding AJ, Norby FL, Ren BH, Ye G, Epstein PN, Ren J. Overexpression of alcohol dehydrogenase exacerbates ethanol-induced contractile defect in cardiac myocytes. Am J Physiol Heart Circ Physiol 2002; 282:H1216-22. [PMID: 11893554 DOI: 10.1152/ajpheart.00780.2001] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Alcoholic cardiomyopathy is characterized by impaired ventricular function although its toxic mechanism is unclear. This study examined the impact of cardiac overexpression of alcohol dehydrogenase (ADH), which oxidizes ethanol into acetaldehyde (ACA), on ethanol-induced cardiac contractile defect. Mechanical and intracellular Ca(2+) properties were evaluated in ventricular myocytes from ADH transgenic and wild-type (FVB) mice. ACA production was assessed by gas chromatography. ADH myocytes exhibited similar mechanical properties but a higher efficiency to convert ACA compared with FVB myocytes. Acute exposure to ethanol depressed cell shortening and intracellular Ca(2+) in the FVB group with maximal inhibitions of 23.3% and 23.4%, respectively. Strikingly, the ethanol-induced depression on cell shortening and intracellular Ca(2+) was significantly augmented in the ADH group, with maximal inhibitions of 43.7% and 40.6%, respectively. Pretreatment with the ADH inhibitor 4-methylpyrazole (4-MP) or the aldehyde dehydrogenase inhibitor cyanamide prevented or augmented the ethanol-induced inhibition, respectively, in the ADH but not the FVB group. The ADH transgene also substantiated the ethanol-induced inhibition of maximal velocity of shortening/relengthening and unmasked an ethanol-induced prolongation of the duration of shortening/relengthening, which was abolished by 4-MP. These data suggest that elevated cardiac ACA exposure due to enhanced ADH expression may play an important role in the development of alcoholic cardiomyopathy.
Collapse
Affiliation(s)
- Jinhong Duan
- Department of Pharmacology, Physiology, and Therapeutics, Grand Forks, North Dakota 58203, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
35
|
Liang Q, Carlson EC, Donthi RV, Kralik PM, Shen X, Epstein PN. Overexpression of metallothionein reduces diabetic cardiomyopathy. Diabetes 2002; 51:174-81. [PMID: 11756338 DOI: 10.2337/diabetes.51.1.174] [Citation(s) in RCA: 140] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Many diabetic patients suffer from cardiomyopathy, even in the absence of vascular disease. This diabetic cardiomyopathy predisposes patients to heart failure and mortality from myocardial infarction. Evidence from animal models suggests that reactive oxygen species play an important role in the development of diabetic cardiomyopathy. Our laboratory previously developed a transgenic mouse model with targeted overexpression of the antioxidant protein metallothionein (MT) in the heart. In this study we used MT-transgenic mice to test whether an antioxidant protein can reduce cardiomyopathy in the OVE26 transgenic model of diabetes. OVE26 diabetic mice exhibited cardiomyopathy characterized by significantly altered mRNA expression, clear morphological abnormalities, and reduced contractility under ischemic conditions. Diabetic hearts appeared to be under oxidative stress because they had significantly elevated oxidized glutathione (GSSG). Diabetic mice with elevated cardiac MT (called OVE26MT mice) were obtained by crossing OVE26 transgenic mice with MT transgenic mice. Hyperglycemia in OVE26MT mice was indistinguishable from hyperglycemia in OVE26 mice. Despite this, the MT transgene significantly reduced cardiomyopathy in diabetic mice: OVE26MT hearts showed more normal levels of mRNA and GSSG. Typically, OVE26MT hearts were found to be morphologically normal, and elevated MT improved the impaired ischemic contractility seen in diabetic hearts. These results demonstrate that cardiomyocyte-specific expression of an antioxidant protein reduces damage to the diabetic heart.
Collapse
Affiliation(s)
- Qiangrong Liang
- Division of Molecular Cardiovascular Biology, University of Cincinnati, Ohio, USA
| | | | | | | | | | | |
Collapse
|
|
36
|
Sato M, Watanabe T, Oshida A, Nagashima A, Miyazaki JI, Kimura M. Usefulness of double gene construct for rapid identification of transgenic mice exhibiting tissue-specific gene expression. Mol Reprod Dev 2001; 60:446-56. [PMID: 11746955 DOI: 10.1002/mrd.1109] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Identification of transgenics still requires PCR and genomic Southern blot hybridization of genomic DNA isolated from tail pieces. Furthermore, identification of transgene-expressing transgenics (hereafter called "expressor") requires mRNA analyses (RT-PCR and Northern blot hybridization) or protein analysis (Western blotting and immunohistochemical staining using specific antibodies). These approaches are often labor-intensive and time-consuming. We developed a technique that simplifies the process of screening expressor transgenics using enhanced green fluorescent protein (EGFP), a noninvasive reporter recently utilized in a variety of organisms, including mice, as a tag. We constructed a MNCE transgene consisting of two expression units, MBP-NCre (termed "MN") and CAG-EGFP (termed "CE"). MN consists of a myelin basic protein (MBP) promoter and NCre gene (Cre gene carrying a nuclear localization signal (NLS) sequence at its 5' end). CE consists of a promoter element, CAG composed of cytomegalovirus (CMV) enhancer and chicken beta-actin promoter, and EGFP cDNA. Of a total of 72 F0 mice obtained after pronuclear injection of MNCE at 1-cell egg stage, 15 were found to express EGFP when the tail, eye, and inner surface of the ear were inspected for EGFP fluorescence under UV illumination at weaning stage. These fluorescent mice were found to possess MNCE and to express NCre mRNA in a brain-specific manner. Mice exhibiting no fluorescence were transgenic or nontransgenic. Mice carrying MNCE, but exhibiting no fluorescence, never expressed NCre mRNA in any organs tested. These findings indicate that (i) direct inspection of the surface of mice for fluorescence under UV illumination enables identification of expressor transgenics without performances of the molecular biological analyses mentioned above, and (ii) systemic promoters such as CAG do not affect the tissue-specificity of a tissue-specific promoter such as MBP promoter, which is located upstream of CAG by approximately 2 kb.
Collapse
MESH Headings
- Actins/genetics
- Animals
- Blotting, Southern
- Brain/metabolism
- Cells, Cultured
- Chickens/genetics
- Cytomegalovirus/genetics
- DNA, Recombinant/genetics
- Enhancer Elements, Genetic/genetics
- Gene Expression
- Gene Expression Profiling/methods
- Genes, Reporter/genetics
- Green Fluorescent Proteins
- Luminescent Proteins/genetics
- Luminescent Proteins/metabolism
- Mice
- Mice, Transgenic
- Myelin Basic Protein/genetics
- Organ Specificity
- Promoter Regions, Genetic/genetics
- RNA, Messenger/analysis
- RNA, Messenger/genetics
- Recombination, Genetic/genetics
- Reverse Transcriptase Polymerase Chain Reaction
- Transfection
- Transgenes/genetics
Collapse
Affiliation(s)
- M Sato
- Molecular Medicine Research Center, The Institute of Medical Sciences, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan.
| | | | | | | | | | | |
Collapse
|
|
37
|
Chen H, Carlson EC, Pellet L, Moritz JT, Epstein PN. Overexpression of metallothionein in pancreatic beta-cells reduces streptozotocin-induced DNA damage and diabetes. Diabetes 2001; 50:2040-6. [PMID: 11522669 DOI: 10.2337/diabetes.50.9.2040] [Citation(s) in RCA: 107] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
The release of reactive oxygen species (ROS) has been proposed as a cause of streptozotocin (STZ)-induced beta-cell damage. This initiates a destructive cascade, consisting of DNA damage, excess activation of the DNA repair enzyme poly(ADP-ribose) polymerase, and depletion of cellular NAD+. Metallothionein (MT) is an inducible antioxidant protein that has been shown to protect DNA from chemical damage in several cell types. Therefore, we examined whether overexpression of MT could protect beta-cell DNA and thereby prevent STZ-induced diabetes. Two lines of transgenic mice were produced with up to a 30-fold elevation in beta-cell MT. Cultured islets from control mice and MT transgenic mice were exposed to STZ. MT was found to decrease STZ-induced islet disruption, DNA breakage, and depletion of NAD+. To assess in vivo protection, transgenic and control mice were injected with STZ. Transgenic mice had significantly reduced hyperglycemia. Ultrastructural examination of islets from STZ-treated mice showed that MT prevented degranulation and cell death. These results demonstrate that MT can reduce diabetes and confirm the DNA damage mechanism of STZ-induced beta-cell death.
Collapse
Affiliation(s)
- H Chen
- Department of Pediatrics, University of Louisville, Louisville, Kentucky 40202, USA
| | | | | | | | | |
Collapse
|
|
38
|
Abstract
Experimental approaches for deciphering the function of human genes rely heavily on our ability to generate mutations in model organisms such as the mouse. However, because recessive mutations are masked by the wild-type allele in the diploid context, conventional mutagenesis and screening is often laborious and costly. Chromosome engineering combines the power of gene targeting in embryonic stem (ES) cells with Cre--loxP technology to create mice that are functionally haploid in discrete portions of the genome. Chromosome deletions, duplications and inversions can be tagged with visible markers, facilitating strain maintenance. These approaches allow for more refined mutagenesis screens that will greatly accelerate functional mouse genomics and generate mammalian models for developmental processes and cancer.
Collapse
Affiliation(s)
- A A Mills
- Cold Spring Harbor Laboratory, 1 BungtownRoad, Cold Spring Harbor, NY 11724, USA.
| | | |
Collapse
|
|
39
|
Jankowsky JL, Slunt HH, Ratovitski T, Jenkins NA, Copeland NG, Borchelt DR. Co-expression of multiple transgenes in mouse CNS: a comparison of strategies. BIOMOLECULAR ENGINEERING 2001; 17:157-65. [PMID: 11337275 DOI: 10.1016/s1389-0344(01)00067-3] [Citation(s) in RCA: 660] [Impact Index Per Article: 27.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The introduction of two transgenes into one animal is increasingly common as transgenic experiments become more sophisticated. In this study we examine two strategies for creating double transgenic founders from a single microinjection. In the first approach, two constructs, each with its own promoter element, were coinjected into the pronucleus. In the second approach, both transgenes were cloned into one vector, separated by an internal ribosomal entry site (IRES), and placed under control of a single promoter. Both strategies save time and increase the percentage of double transgenic offspring over the standard method of mating single transgenic lines. However, despite high transgene copy numbers, the bicistronic lines did not show robust expression of either protein. Copy number and protein expression correlated much better in the coinjected lines, with expression levels in one line approaching that observed in some of our best single transgenic controls. Thus we recommend coinjection of individual plasmids for the generation of multiply transgenic founders.
Collapse
Affiliation(s)
- J L Jankowsky
- Department of Pathology, Johns Hopkins School of Medicine, 720 Rutland Ave., 558 Ross Research Building, Baltimore, MD 21205, USA
| | | | | | | | | | | |
Collapse
|
|
40
|
Colitz CM, Malarkey DE, Woychik RP, Wilkinson JE. Persistent hyperplastic tunica vasculosa lentis and persistent hyperplastic primary vitreous in transgenic line TgN3261Rpw. Vet Pathol 2000; 37:422-7. [PMID: 11055865 DOI: 10.1354/vp.37-5-422] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Persistent hyperplastic tunica vasculosa lentis and persistent hyperplastic primary vitreous are congenital ocular anomalies that can lead to cataract formation. A line of insertional mutant mice, TgN3261Rpw, generated at the Oak Ridge National Laboratory in a large-scale insertional mutagenesis program was found to have a low incidence (8/243; 3.29%) of multiple developmental ocular abnormalities. The ocular abnormalities include persistent hyperplastic primary vitreous, persistent hyperplastic tunica vasculosa lentis, failure of cleavage of the anterior segment, retrolental fibrovascular membrane, posterior polar cataract, and detached retina. This transgenic mouse line provides an ontogenetic model because of the high degree of similarity of this entity in humans, dogs, and mice.
Collapse
Affiliation(s)
- C M Colitz
- Department of Companion Animal and Special Species Medicine, North Carolina State University, College of Veterinary Medicine, Raleigh, USA
| | | | | | | |
Collapse
|
|
41
|
Lu W, Phillips CL, Killen PD, Hlaing T, Harrison WR, Elder FF, Miner JH, Overbeek PA, Meisler MH. Insertional mutation of the collagen genes Col4a3 and Col4a4 in a mouse model of Alport syndrome. Genomics 1999; 61:113-24. [PMID: 10534397 DOI: 10.1006/geno.1999.5943] [Citation(s) in RCA: 46] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Mice homozygous for the transgenic insertion in line OVE250 exhibit severe progressive glomerulonephritis. Ultrastructural changes in the glomerular basement membrane (GBM) at 2 weeks of age resemble those in Alport syndrome. The transgenic insertion site was mapped by FISH to mouse chromosome 1 close to Pax3. Genetic and molecular analyses identified a deletion of genomic DNA at the transgene insertion site. Exons 1 through 12 of the collagen IV gene Col4a4, exons 1 and 2 of the adjacent Col4a3 gene, and the intergenic promoter region are deleted. Transcripts of Col4a3 and Col4a4 are undetectable in mutant kidney, and both proteins are missing from the GBM. Persistent cellular proliferation in mutant kidneys suggests that interaction with the extracellular matrix may be important for cell maturation. Evolutionarily conserved sequence elements in the promoter regions of human and mouse Col4a3 and Col4a4 include a 19-bp element that was tandemly duplicated in the human lineage and a CTC box element common to several genes encoding extracellular matrix proteins. This new animal model of Alport syndrome, Col4Delta3-4, lacks both alpha3 and alpha4 chains of collagen IV and exhibits an earlier disease onset than mice lacking alpha3 only.
Collapse
Affiliation(s)
- W Lu
- Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, 48109, USA
| | | | | | | | | | | | | | | | | |
Collapse
|
|
42
|
Zheng B, Mills AA, Bradley A. A system for rapid generation of coat color-tagged knockouts and defined chromosomal rearrangements in mice. Nucleic Acids Res 1999; 27:2354-60. [PMID: 10325425 PMCID: PMC148802 DOI: 10.1093/nar/27.11.2354] [Citation(s) in RCA: 97] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Gene targeting in mouse embryonic stem (ES) cells can be used to generate single gene mutations or defined multi-megabase chromosomal rearrangements when applied with the Cre- loxP recombination system. While single knockouts are essential for uncovering functions of cloned genes, chromosomal rearrangements are great genetic tools for mapping, mutagenesis screens and functional genomics. The conventional approach to generate mice with targeted alterations of the genome requires extensive molecular cloning to build targeting vectors and DNA-based genotyping for stock maintenance. Here we describe the design and construction of a two-library system to facilitate high throughput gene targeting and chromo-somal engineering. The unique feature of these libraries is that once a clone is isolated, it is essentially ready to be used for insertional targeting in ES cells. The two libraries each bear a complementary set of genetic markers tailored so that the vector can be used for Cre- loxP -based chromosome engineering as well as single knockouts. By incorporating mouse coat color markers into the vectors, we illustrate a widely applicable method for stock maintenance of ES cell-derived mice with single gene knockouts or more extensive chromosomal rearrangements.
Collapse
Affiliation(s)
- B Zheng
- Department of Molecular and Human Genetics and Howard Hughes Medical Institute, Baylor College of Medicine,1 Baylor Plaza, Houston, TX 77030, USA
| | | | | |
Collapse
|
|
43
|
Ikawa M, Yamada S, Nakanishi T, Okabe M. Green fluorescent protein (GFP) as a vital marker in mammals. Curr Top Dev Biol 1999; 44:1-20. [PMID: 9891875 DOI: 10.1016/s0070-2153(08)60465-2] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Affiliation(s)
- M Ikawa
- Program for Promotion of Basic Research Activities for Innovative Biosciences, Osaka University, Suita, Japan
| | | | | | | |
Collapse
|
|
44
|
Sallinen J, Haapalinna A, Viitamaa T, Kobilka BK, Scheinin M. D-amphetamine and L-5-hydroxytryptophan-induced behaviours in mice with genetically-altered expression of the alpha2C-adrenergic receptor subtype. Neuroscience 1998; 86:959-65. [PMID: 9692731 DOI: 10.1016/s0306-4522(98)00100-6] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Three human and mouse genes encoding alpha2-adrenoceptor subtypes (alpha2A, alpha2B, and alpha2C) have been cloned. The alpha2C-adrenoceptor is the most abundant alpha2-adrenoceptor subtype in the striatum and modulates metabolism of both dopamine and serotonin. To investigate the possible involvement of the alpha2C-adrenoceptor subtype in behaviours regulated by dopamine and serotonin, two strains of genetically-engineered mice were examined. One had a targeted inactivation of the alpha2C-adrenoceptor gene, and the other had tissue-specific over-expression of alpha2C-adrenoceptors. The locomotor activity of the mice was evaluated after stimulation with D-amphetamine, and the behavioural serotonin syndrome and head twitches were investigated after L-5-hydroxytryptophan treatment. In addition, the effects of D-amphetamine and L-5-hydroxytryptophan were studied after pretreatment with dexmedetomidine, a subtype-nonselective alpha2-adrenoceptor agonist. The lack of alpha2C-adrenoceptor expression increased and the over-expression of alpha2C-adrenoceptors decreased the response to D-amphetamine stimulation. The effect of alpha2C-adrenoceptor gene inactivation was more prominent in D-amphetamine-treated males than in females. Dexmedetomidine inhibited D-amphetamine-induced hyperlocomotion and the L-5-hydroxytryptophan-induced serotonin syndrome, but the inhibition was attenuated in mice lacking alpha2C-adrenoceptors. However, the head twitches induced by L-5-hydroxytryptophan were effectively inhibited by dexmedetomidine in all studied mice, which suggests that alpha2A-adrenoceptors mediate the inhibition of the head twitch response. The results lend further support to the proposed existence of functionally distinct alpha2-adrenoceptor subtypes that can serve as new and specific therapeutic targets in various neuropsychiatric diseases.
Collapse
Affiliation(s)
- J Sallinen
- Department of Pharmacology and Clinical Pharmacology, University of Turku, Finland
| | | | | | | | | |
Collapse
|
|
45
|
Goldman MA, Reeves PS, Wirth CM, Zupko WJ, Wong MA, Edelhoff S, Disteche CM. Comparative methylation analysis of murine transgenes that undergo or escape X-chromosome inactivation. Chromosome Res 1998; 6:397-404. [PMID: 9872669 DOI: 10.1023/a:1009229423535] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
We analyzed an X-linked metallothionein-vasopressin (MTVP) fusion transgene that undergoes X-chromosome inactivation (X inactivation) and an X-linked transferrin (TFN) transgene that escapes X inactivation with respect to methylation in the 5' regulatory regions. The MTVP transgene promoter region is unmethylated when the transgene is on the active X chromosome and methylated when on the inactive X chromosome. Interestingly, the MTVP transgene is not detectably transcribed from the male X chromosome, although it is unmethylated, consistent with its availability for transcription. The TFN transgene promoter region is hypomethylated on both the active and inactive X chromosomes, consistent with its expression from both chromosomes. The TFN and MTVP transgenes have been mapped to chromosomal regions D and C, respectively, by fluorescence in situ hybridization. These observations are discussed in the context of our understanding of the role of DNA methylation in the spread and maintenance of X-chromosome inactivation.
Collapse
Affiliation(s)
- M A Goldman
- Department of Biology, San Francisco State University, CA 94132-1722, USA.
| | | | | | | | | | | | | |
Collapse
|
|
46
|
Woychik RP, Klebig ML, Justice MJ, Magnuson TR, Avner ED, Avrer ED. Functional genomics in the post-genome era. Mutat Res 1998; 400:3-14. [PMID: 9685569 DOI: 10.1016/s0027-5107(98)00023-2] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
As the biomedical research community enters the post-genome era, studying gene expression patterns and phenotypes in model organisms will be an important part of analyzing the role of genes in human health and disease. New technologies involving DNA chips will improve the ability to evaluate the differential expression of a large number of genes simultaneously. Also, new approaches for generating mutations in mice will significantly decrease the cost and increase the rate of generating mutant lines that model human disease.
Collapse
Affiliation(s)
- R P Woychik
- Department of Pediatrics, Case Western Reserve University, Cleveland, OH 44106, USA.
| | | | | | | | | | | |
Collapse
|
|
47
|
Adrenergic alpha2C-receptors modulate the acoustic startle reflex, prepulse inhibition, and aggression in mice. J Neurosci 1998. [PMID: 9526020 DOI: 10.1523/jneurosci.18-08-03035.1998] [Citation(s) in RCA: 130] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
Studies on animal models of stress, anxiety, aggression, and sensorimotor gating have linked specific monoamine neurotransmitter abnormalities to the cognitive and behavioral disturbances associated with many affective neuropsychiatric disorders. Although alpha2-adrenoceptors (alpha2-ARs) have been suggested to have a modulatory role in these disorders, the specific roles of each alpha2-AR subtype (alpha2A, alpha2B, and alpha2C) are largely unknown. The restricted availability of relevant animal models and the lack of subtype-selective alpha2-AR drugs have precluded detailed studies in this area. Therefore, transgenic mice were used to study the possible role of the alpha2C-AR subtype in two well established behavioral paradigms: prepulse inhibition (PPI) of the startle reflex and isolation-induced aggression. The alpha2C-AR-altered mice appear grossly normal, but subtle changes have been observed in their brain dopamine (DA) and serotonin (5-HT) metabolism. In this study, the mice with targeted inactivation of the gene encoding alpha2C-ARs (alpha2C-KO) had enhanced startle responses, diminished PPI, and shortened attack latency in the isolation-aggression test, whereas tissue-specific overexpression of alpha2C-ARs (alpha2C-OE) was associated with opposite effects. Correlation analyses suggested that both the magnitude of the startle response and its relative PPI (PPI%) were modulated by the mutations. In addition, the differences in PPI, observed between drug-naive alpha2C-OE mice and their wild-type controls, were abolished by treatment with a subtype nonselective alpha2-agonist and antagonist. Thus, drugs acting via alpha2C-ARs might have therapeutic value in disorders associated with enhanced startle responses and sensorimotor gating deficits, such as schizophrenia, attention deficit disorder, post-traumatic stress disorder, and drug withdrawal.
Collapse
|
|
48
|
Justice MJ, Zheng B, Woychik RP, Bradley A. Using targeted large deletions and high-efficiency N-ethyl-N-nitrosourea mutagenesis for functional analyses of the mammalian genome. Methods 1997; 13:423-36. [PMID: 9480786 DOI: 10.1006/meth.1997.0548] [Citation(s) in RCA: 61] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The Human Genome Project has generated nucleotide sequences from an estimated 80,000 to 100,000 genes, only a small fraction of which have a known role. Nucleotide sequence information alone is insufficient to predict gene function. One of the most powerful ways of revealing gene function, as demonstrated in bacteria, worms, yeast, and flies, is to generate mutations and characterize them at both the phenotypic and the molecular levels. Given the physiological and anatomical parallels between mouse and human, genotype-phenotype relationships established in mice can be extrapolated to human syndromes. A new method is described for functional genetic analyses in the mouse that uses loxP/Cre engineering to generate coat color-tagged large deletions. The haploid regions can then be dissected by mutagenesis with N-ethyl-N-nitrosourea in phenotype-driven screens to obtain functional information on genes in any desired region of the mouse genome.
Collapse
Affiliation(s)
- M J Justice
- Life Sciences Division, Oak Ridge National Laboratory, Tennessee 37830, USA
| | | | | | | |
Collapse
|
|
49
|
Brenin DR, Talamonti MS, Iannaccone PM. Transgenic technology: an overview of approaches useful in surgical research. Surg Oncol 1997; 6:99-110. [PMID: 9436656 DOI: 10.1016/s0960-7404(97)00010-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Advances in transgenic science have created powerful tools for the investigation of both genetic and protein regulatory systems. Recently, transgenic animals have been utilized in several vascular and transplantation research laboratories. The ability to specifically mutate genes important in oncologic and cardiovascular research is leading to a greater understanding of the role of gene and protein regulatory systems in cancer and cardiovascular disease. The expanding use of transgenic animals will undoubtedly increase our insight into complex problems in surgical research. This review briefly describes the various techniques utilized to create transgenic animals including: transgene design, gene-transfer utilizing transfection, microinjection and retroviral infection, as well as the use of embryonic stem cells, and methods for screening transgenic offspring.
Collapse
Affiliation(s)
- D R Brenin
- Department of Surgery, Northwestern University Medical School, Chicago, IL 60611, USA
| | | | | |
Collapse
|
|
50
|
Hein L, Stevens ME, Barsh GS, Pratt RE, Kobilka BK, Dzau VJ. Overexpression of angiotensin AT1 receptor transgene in the mouse myocardium produces a lethal phenotype associated with myocyte hyperplasia and heart block. Proc Natl Acad Sci U S A 1997; 94:6391-6. [PMID: 9177228 PMCID: PMC21060 DOI: 10.1073/pnas.94.12.6391] [Citation(s) in RCA: 148] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Previous studies have suggested that angiotensin II (Ang II) modulates cardiac contractility, rhythm, metabolism, and structure. However, it is unclear whether the cardiac effects are due to direct actions of Ang II on the myocardium or if they are due to secondary effects mediated through the hemodynamic actions of Ang II. In this study, we used the alpha-myosin heavy chain (alphaMHC) promoter to generate transgenic mice overexpressing angiotensin II type 1 (AT1a) receptor selectively in cardiac myocytes. The specificity of transgene expression in the transgenic offspring was confirmed by radioligand binding studies and reverse transcription-PCR. The offspring displayed massive atrial enlargement with myocyte hyperplasia at birth, developed significant bradycardia with heart block, and died within the first weeks after birth. Thus, direct activation of AT1 receptor signaling in cardiac myocytes in vivo is sufficient to induce cardiac myocyte growth and alter electrical conduction.
Collapse
Affiliation(s)
- L Hein
- Falk Cardiovascular Research Center and Department of Medicine, 300 Pasteur Drive, Stanford University School of Medicine, Stanford, CA 94305, USA
| | | | | | | | | | | |
Collapse
|