Original Article
Copyright ©2010 Baishideng Publishing Group Co.
World J Stem Cells. Dec 26, 2010; 2(6): 127-140
Published online Dec 26, 2010. doi: 10.4252/wjsc.v2.i6.127
Table 1 Some characteristics of R1 and HM-1 embryonic stem cells and fluorescence activated cell sorting analysis
Cell lineR1HM-1
Genetic background(129X1/SvJ x 129S1/Sv) F1 (+/p+tyr-cKitlSl-J/+)1129/Ola2
Culture conditionsDMEM3 + 15%FBS + 1000 U ESGRO mLIFDMEM3 + 15%FBS + 1000 U ESGRO mLIF
Cell doubling (in our hands)14-16 h12-14 h
Pluripotency
Germline competence4++
In vitro differentiation to form three germ layers5++
Karyotype (FISH)XY (often XO)XY more stabile
Euploidy (hoechst staining)78% ± 4.4%71% ± 5.3%
Telomerase (PCR)++
FACS analysis (mean ± SE)
Pou5f1 (FACS)90.6% ± 5.8%88.8% ± 5.7%
Nanog (FACS)72.5% ± 6.2%68.7% ± 2.8%
SSEA-1 (FACS)58.8% ± 4.1%65.1% ± 3.1%
Sox2 (FACS)97.4% ± 1.2%98.8% ± 0.2%
ALP (enzyme assay)++
Table 2 Comparative targeting efficiencies of R1 and HM-1 mouse embryonic stem cells
ParametersCell line (passage number)
R1 (p12)
HM-1 (p21)
Amaxa
BioRadAmaxa
BioRad
Electroporation programA231A241A301A231A241A301
Cell number3 × 1063 × 1073 × 1063 × 107
DNA concentration (μg)510510
Number of selected clones12019216835683616816824384744
Positive clones after 1st PCR screen3101115395401524
Percentage (%)2.55.26.54.24.72.92.403.93.2
Positive clones after 2nd PCR screen3101115395401524
Homologous recombination confirmed by Southern blot analysis
Clone number3910729440514
Efficiency (%)2.54.7623.52.42.401.31.9
Transfection efficiency
TransientNo data on Amaxa system91% ± 6.1%No data on Amaxa system75% ± 4.2%
StableNo data on Amaxa system34% ± 2.9%No data on Amaxa system25% ± 5.3%
Table 3 Differentially regulated transcripts between R1 and HM-1 embryonic stem cells
Lion IDGene symbolGene nameLocus IDUnigene IDReference sequenceFold change
Chrom location
MinAverStdv
Up regulated in HM-1 compared to the R1 embryonic stem cells
MG-4-li7Rps15aRibosomal protein S15a267019Mm.288212NM_1706691.441.50.087 F1
MG-3-6b3HSPa9aHeat shock protein 9a15526Mm.209419NM_0104811.41.480.0518 15cM
MG-14-78c20B930046C15RikRIKEN cDNA B930046C15Rik gene544998Mm.327147XM_9870631.391.530.114 A1
MG-13-43a6Lefty1Left right determination factor13590Mm.378911NM_0100941.331.50.141 F
MG-3-1 1116LOC544988Hypothetical protein LOC544988544988Mm.350858NM_0010247121.291.40.0814 A1
MG-3-32g7Nsmce1Non.SMC element 1 homolog67711Mm.4467NM_0263301.281.430.17 F3
MG-11-2g16Glo1Glyoxylase 1109801Mm.261984NM_0253741.261.330.0517 16.0 cM
MG-3-223f10EST1.262.351.26
Down regulated in HM-1 compared to the R1 embryonic stem cells
MG-15-102f2Dab2Disabled homolog 2 drosophila13132Mm.240830NM_0010087021.992.130.1315 6.7cM
MG-12-140m7Spink3Serine protease inhibitor Kazal-type 3 precursor20730Mm.272NM_0092581.922.10.1418 B3
MG-3-251i14Sparcsecreted acidic cystein rich glycoprotein20692Mm.291442NM_0092421.862.050.1311 29.9 cM
MG-15-55i8AmotAngiomotin27494Mm.100068NM_1533191.761.90.14X F2
MG-8-118g22Krt2-8Keratin complex 2 basic gene 816691Mm.358618NM_0311701.751.930.1315 58.86cM
MG-13-5n3CryabCrystallin, α B12955Mm.178NM_0099641.71.780.19 29.0cM
MG-15-261m9BC024814cDNA sequence BC024814239706Mm.214953NM_1462471.691.880.1516 A1
MG-8-17b12Prph1Peripherin 119132Mm.2477NM_0136391.661.980.2515 55.5 cM
Table 3b. cont.Gpc4Glypican-4 precursor (K-glypican)14735Mm.1528NM_0081501.622.10.37X 16.0 cM
MG-8-34a20
MG-13-91a5Serpinh1Serine (Cystein) proteinase inhibitor, clade H, member 112406Mm.22708NM_0098251.561.630.057 E1
MG-14-101o20CtgfConnective tissue growth factor precursor14219Mm.1810NM_0102171.561.730.110 17 cM
MG-12-197o17PodxlPodocalyxin like protein 1 precursor27205Mm.89918NM_0137231.461.580.156 10.0 cM
MG-6-75c23Lamc1Laminin gamma 1226519Mm.1249NM_0106831.451.50.081 81.1 cM
MG-8-118f19Anxa2Annexin 212306Mm.238343NM_0075851.451.50.089 37.0 cM
MG-16-54i39630046K23RikRIKEN cDNA 9630046K23 gene224143Mm.284366NM_1723801.441.580.1316 B3
MG-6-2i22Timp2tissue inhibitor of metalloproteinase 221858Mm.206505NM_0115941.421.50.0811 72.0 cM
MG-13-1c9S100a10Calpactin I light chain20194Mm.1NM_0091121.411.480.053 41.7 cM
MG-14-79j4Lhfp12Lipoma HMGIC fusion partner-like 2, mRNA218454Mm.316553NM_1725891.411.580.2113 C3-D1
MG-3-38p22CtslCathepsin L precursor13039Mm.930NM_0099841.381.630.1513 30.0 cM
MG-3-91d9Kcnh2Potassium voltage-gated channel, sub family H (eagralated), member 216511Mm.6539AC1130551.381.530.15
MG-3-45k24GsnGelsoin227753Mm.21109NM_1461201.371.480.152 24.5 cM
MG-6-31m16Clec21C-type lectin domain family, member 1381758Mm.349066XM_3557531.371.406 B1
MG-13-2e2ESTESTCR5177951.361.480.1
MG-3-171n1Iap1-3Intracisternal A particles, Eya 1 linked15601AF0975461.351.50.081 10.4 cM
MG-3-85d19SfrsiSplicing factor, arginine/serine-rich (ASF/SFZ)Mm.45645CR5181311.342.050.8911
MG-12-195b2Timp3Metalloproteinase inhibitor 3 precursor (TIMP3) tissue inhibitor21859Mm.4871NM_0115951.331.450.1310 47.0 cM
MG-14-58h24Col8a1Procollagen type VIII, α 112837Mm.370175BC0582811.331.450.116 C1.1
MG-3-23b6Tpm1Tropomyosin 1 α22003Mm.121878M224791.321.380.059 40.0 cM
MG-13-1k15S100a4S100 calcium binding protein A420198Mm.3925BC0512141.311.450.133 43.6 cM
Table 3b. cont.S100a1S100 calcium binding protein A120193Mm.24662NM_0113091.311.350.063 43.6 cM
MG-3-72e5
MG-68-98m4CtszCathepsin Z precursor64138Mm.156919NM_0223251.311.40.082 103.5 cM
MG-13-117j9AhnakAHNAK nucleoprotein (desmoyokin), mRNA66395Mm.203866BC0068921.291.40.0819 A
MG-15-217f23A930026I22RikRIKEN cDNA A930026I22 gene77970Mm.259790BX6323871.281.380.0519 C3
MG-6-30n6S100a6S100 calcium binding protein A6 (Calcyclin)20200Mm.100144NM_0113131.281.450.13 43.6 cM
MG-6-30o19Clstn3calsyntenin 3232370Mm.193701NM_1535081.281.350.066 F2
MG-6-31b18Camkk2Calcium/calmodulin-dependent protein kinase kinase 2 β207565Mm.289237NM_1453581.281.305 F
MG-12-3f11S100gS100 calcium binding protein G12309Mm.6891NM_0097891.271.30X F4
MG-6-1g19My16myosin, light polypeptide 6, alkali, smooth muscle and non-muscle17904Mm.337074NM_0108601.271.350.0610
MG-6-31b244933433P14RikRIKEN cDNA 4933433P14 gene66787Mm.248019BC0314941.271.40.0812 E
MG-12-42l4Cotl1Coactosin-like 1 (Dictyostelium)72042Mm.141741XM_4892501.261.450.138 E1
MG-3-254m20A430065p19Riken A430065P19 gene329421Mm.99648NM_1773761.261.450.132 C2
MG-3-37p2Qscn6Quiescin Q6104009Mm.27035NM_0010249451.261.380.051 G3
MG-6-31h10Map3k11mitogen activated protein kinase kinase kinase 1126403Mm.185020NM-0220121.261.330.0519 0.5 cM
MG-6-82c15DbpD site albumin promoter binding protein13170Mm.378235NM_0169741.261.307 23.0 cM
MG-8-17n2Stard9START domain containing 9211824Mm.246506XM_6198011.261.380.052 F1
MG-14-95h3Ggta1Glycoprotein galactosyltransferase α 114594Mm.281124NM_0102831.251.380.152 25.0 cM
MG-3-6912AC116589AC1165891.251.40.0813
Table 4 Primer sequences and polymerase chain reaction conditions of genes selected for real time polymerase chain reaction assays
Gene symbolGene namePrimer sequence (5' to 3')Product size (bp)Annealing T°
S100a4S100 calcium binding protein A4For-TCAAGCTGAACAAGACAGAGC13156
Rev-ACTTCATTGTCCCTGTTGCTG
S100a6S100 calcium binding protein A6For-TCCACAAGTACTCTGGCAAGG13856
Rev-GGTCCAGATCATCCATCAGC
S100a10S100 calcium binding protein A10For-AGCTCTTCCAAGGACTGCTG13860
Rev-TTTGTCAAGTGGTCTTTGTCG
Camkk2Calcium/calmodulin-dependent protein kinase kinase2, βFor-GTCACACCACGTCTCCATTAC14456
Rev-ACTTTCATTGCGTAATAAGTATTGTC
Tpm1Tropomyosin 1 αFor-ATGCCCGTGTTCCTTAAAGC15759
Rev-CCCTGACATGGAGAACTGGG
SparcSecreted acidic cystein rich glycoproteinFor-ATCCCCATGGAACATTGCAC12259
Rev-TCCTTGTTGATGTCCTGCTCC
TrhThyrotropin releasing hormoneFor-GGTTCTTCCACGCCTCCTAAG13559
Rev-AACCTTGGAGGATGCGCTG
Lefty1Left right determination factor 1For-GCAAACCAAGGACAGAATCCC10859
Rev-TGTTTGCCCAACTTGTGGC
P2rx1Purinergic receptor P2X, ligand-gated ion channel 1For-GTCTCCAGGCTTCAACTTC15656
Rev-GAGCCGATGGTAGTCATAGT
P2rx2Purinergic receptor P2X, ligand-gated ion channel 2For-GTAGAGCAAGCAGGAGAGA8960
Rev-AGACAAGTCCAGGTCACAG
P2rx3Purinergic receptor P2X, ligand-gated ion channel 3For-CCCGCTAAGACCTGAATCT14260
Rev-AGTCCCTGGTATGTGGTAGG
P2rx4Purinergic receptor P2X, ligand-gated ion channel 4For-CTGTGTGACGTCATAGTCC8560
Rev-GCTCGTAGTCTTCCACATAC
P2rx5Purinergic receptor P2X, ligand-gated ion channel 5For-GCTTTCTTCTGTGACCTG9960
Rev-ATCTTCCTCCTTCTGACC
P2rx6Purinergic receptor P2X, ligand-gated ion channel 6For-CCCAAAGACGACTACCAA8660
Rev-CAAACCACTCCTAGGACACT
P2rx7Purinergic receptor P2X, ligand-gated ion channel 7For-GGAAGTTAACCGTTCCTG9860
Rev-TGGGCTAGACCTACTTCC
P2ry1Purinergic receptor P2Y, G-protein coupled 1For-GGTCTAGCAAGTCTCAACAG12160
Rev-GTAAATTGGCCTCACTCC
P2ry2Purinergic receptor P2Y, G-protein coupled 2For-CCAAGCATGGAGAGGAGT15660
Rev-GAATTGCTTGCACCACAG
P2ry4Purinergic receptor P2Y, G-protein coupled 4For-ACTAACTGCAGGCAGAGG17060
Rev-CCAGCAAAGAGTACTGAGG
P2ry5Purinergic receptor P2Y, G-protein coupled 5For-CATGTACCCGATCACTCTC13660
Rev-GAACCTGGAGTCACTTCTTC
P2ry6Purinergic receptor P2Y, G-protein coupled 6For-GAGTTCTGCGTGTGTGTG17260
Rev-GTCAGCCTTTCCTATGCT
P2ry10Purinergic receptor P2Y, G-protein coupled 10For-GACATTTGGTATGCAGGCAAG8256
Rev-TGGTCCCTTCCTCTTCCTTAGT
P2ry12Purinergic receptor P2Y, G-protein coupled 12For-TGCTGAGGTGCTCAAACTCTAC8456
Rev-GGGTCTCTTCGCTTGGTTC
P2ry13Purinergic receptor P2Y, G-protein coupled 13For-AACAGAGCACCAGAAGAGAG11960
Rev-AGGATGCAGATGCTGTTG
P2ry14Purinergic receptor P2Y, G-protein coupled 14For-CACAAAGAGTCAGACGGAAGG9660
Rev-ACATTGGCAGCCGAGAGTAG
Bdkrb1Bradykinin receptor, β 1For-AGTCGTCCCTGATCTGAA8556
Rev-GTTCAACTCCACCATCCT
Bdkrb2Bradykinin receptor, β 2For-CACGCAGATCAGTTCCTAC9960
Rev-ACCTCTCGGGACTTCTTC
Hrh1Histamine receptor H1For-GGGCTACATCAACTCCAC15360
Rev-CCCTCTTGGACATCAGAC
Hrh2Histamine receptor H2For-CCTCTCCTTCCTCTCTATTC11060
Rev-CCACCAGTCCATATACCTC
Hrh3Histamine receptor H3For-ACAGTCAGCAGGAGGGAGAG13556
Rev-TGTCTTCACATTGGCAGAGG
Hrh4Histamine receptor H4For-CTTGGAAGAACAGCACGAAC10656
Rev-GAGATGACAGGAAGCAGGAA
Ryr1Ryanodine receptor 1, skeletal muscleFor-ACTTTGTACCCTGTCCTGTG13260
Rev-CATAGGTCCATCCTTGCTC
Ryr2Ryanodine receptor 2, cardiac muscleFor-GGATGAATGTCTCACTGTCC14760
Rev-CTTATGTGGCTTCCACTCC
Ryr3Ryanodine receptor 3For-CAGGTATCTTGGAGGTCTTG11160
Rev-GCCCATGCTTATCCAGTAG
Itpr1Inositol 1,4,5-triphosphate receptor 1For-GGTTGATGACCGTTGTGTTG12160
Rev-GAACTGTTTCTGTGCGGAGT
Itpr2Inositol 1,4,5-triphosphate receptor 2For-GTGAGGATGGCATAGAAAG16560
Rev-AGAAGAACAGGAGGTCGTAG
Itpr3Inositol 1,4,5-triphosphate receptor 3For-GGCTTCATCAGCACTTTGG9660
Rev-GCAATCTCGGAACTTCTTGG
H2afzH2A histone family, member ZFor-ACAGCGCAGCCATCCTGGAGTA20260
Rev-TTCCCGATCAGCGATTTGTGGA
PpiaPeptidylprolyl isomerase AFor-CGCGTCTCCTTCGAGCTGTTTG15060
Rev-TGTAAAGTCACCACCCTGGCACAT