Gene targeting and Calcium handling efficiencies in mouse embryonic stem cell lines

Table 1 Some characteristics of R1 and HM-1 embryonic stem cells and fluorescence activated cell sorting analysis

Cell line	R1	HM-1
Genetic background	(129X1/SvJ x 129S1/Sv) F1 (+/p+tyr-cKitlSl-J/+)1	129/Ola2
Culture conditions	DMEM3 + 15%FBS + 1000 U ESGRO mLIF	DMEM3 + 15%FBS + 1000 U ESGRO mLIF
Cell doubling (in our hands)	14-16 h	12-14 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)	+	+

¹http://www.mshri.on.ca/nagy;

²HPRT (hypoxanthine phosphoribosyl transferase)-deficient mice derived;

³DMEM media: DMEM (4.5 g/L glucose) + 2 mmol glutamine + 0.1 mmol MEM non-essential amino-acids + 0.1 β-mercaptoethanol + 1 mmol sodium pyruvate + penicillin and streptomycin;

⁴Based on the literature;

⁵Spontaneous and induced (DMSO for cardiac and RA for neuronal) differentiation was tested. PCR: Polymerase chain reaction; FACS: Fluorescence activated cell sorting.

Table 2 Comparative targeting efficiencies of R1 and HM-1 mouse embryonic stem cells

Parameters	Cell line (passage number)
	R1 (p12)					HM-1 (p21)
	Amaxa			BioRad	∑	Amaxa			BioRad	∑
Electroporation program	A231	A241	A301			A231	A241	A301
Cell number	3 × 106			3 × 107			3 × 106			3 × 107
DNA concentration (μg)	5			10		5			10
Number of selected clones	120	192	168	356	836	168	168	24	384	744
Positive clones after 1st PCR screen	3	10	11	15	39	5	4	0	15	24
Percentage (%)	2.5	5.2	6.5	4.2	4.7	2.9	2.4	0	3.9	3.2
Positive clones after 2nd PCR screen	3	10	11	15	39	5	4	0	15	24
Homologous recombination confirmed by Southern blot analysis
Clone number	3	9	10	7	29	4	4	0	5	14
Efficiency (%)	2.5	4.7	6	2	3.5	2.4	2.4	0	1.3	1.9
Transfection efficiency
Transient	No data on Amaxa system			91% ± 6.1%		No data on Amaxa system			75% ± 4.2%
Stable	No data on Amaxa system			34% ± 2.9%		No data on Amaxa system			25% ± 5.3%

¹Amaxa program numbers. PCR: Polymerase chain reaction.

Table 3 Differentially regulated transcripts between R1 and HM-1 embryonic stem cells

Lion ID	Gene symbol	Gene name	Locus ID	Unigene ID	Reference sequence	Fold change			Chrom location
Min						Aver	Stdv
Up regulated in HM-1 compared to the R1 embryonic stem cells
MG-4-li7	Rps15a	Ribosomal protein S15a	267019	Mm.288212	NM_170669	1.44	1.5	0.08	7 F1
MG-3-6b3	HSPa9a	Heat shock protein 9a	15526	Mm.209419	NM_010481	1.4	1.48	0.05	18 15cM
MG-14-78c20	B930046C15Rik	RIKEN cDNA B930046C15Rik gene	544998	Mm.327147	XM_987063	1.39	1.53	0.1	14 A1
MG-13-43a6	Lefty1	Left right determination factor	13590	Mm.378911	NM_010094	1.33	1.5	0.14	1 F
MG-3-1 1116	LOC544988	Hypothetical protein LOC544988	544988	Mm.350858	NM_001024712	1.29	1.4	0.08	14 A1
MG-3-32g7	Nsmce1	Non.SMC element 1 homolog	67711	Mm.4467	NM_026330	1.28	1.43	0.1	7 F3
MG-11-2g16	Glo1	Glyoxylase 1	109801	Mm.261984	NM_025374	1.26	1.33	0.05	17 16.0 cM
MG-3-223f10	EST					1.26	2.35	1.26
Down regulated in HM-1 compared to the R1 embryonic stem cells
MG-15-102f2	Dab2	Disabled homolog 2 drosophila	13132	Mm.240830	NM_001008702	1.99	2.13	0.13	15 6.7cM
MG-12-140m7	Spink3	Serine protease inhibitor Kazal-type 3 precursor	20730	Mm.272	NM_009258	1.92	2.1	0.14	18 B3
MG-3-251i14	Sparc	secreted acidic cystein rich glycoprotein	20692	Mm.291442	NM_009242	1.86	2.05	0.13	11 29.9 cM
MG-15-55i8	Amot	Angiomotin	27494	Mm.100068	NM_153319	1.76	1.9	0.14	X F2
MG-8-118g22	Krt2-8	Keratin complex 2 basic gene 8	16691	Mm.358618	NM_031170	1.75	1.93	0.13	15 58.86cM
MG-13-5n3	Cryab	Crystallin, α B	12955	Mm.178	NM_009964	1.7	1.78	0.1	9 29.0cM
MG-15-261m9	BC024814	cDNA sequence BC024814	239706	Mm.214953	NM_146247	1.69	1.88	0.15	16 A1
MG-8-17b12	Prph1	Peripherin 1	19132	Mm.2477	NM_013639	1.66	1.98	0.25	15 55.5 cM
Table 3b. cont.	Gpc4	Glypican-4 precursor (K-glypican)	14735	Mm.1528	NM_008150	1.62	2.1	0.37	X 16.0 cM
MG-8-34a20
MG-13-91a5	Serpinh1	Serine (Cystein) proteinase inhibitor, clade H, member 1	12406	Mm.22708	NM_009825	1.56	1.63	0.05	7 E1
MG-14-101o20	Ctgf	Connective tissue growth factor precursor	14219	Mm.1810	NM_010217	1.56	1.73	0.1	10 17 cM
MG-12-197o17	Podxl	Podocalyxin like protein 1 precursor	27205	Mm.89918	NM_013723	1.46	1.58	0.15	6 10.0 cM
MG-6-75c23	Lamc1	Laminin gamma 1	226519	Mm.1249	NM_010683	1.45	1.5	0.08	1 81.1 cM
MG-8-118f19	Anxa2	Annexin 2	12306	Mm.238343	NM_007585	1.45	1.5	0.08	9 37.0 cM
MG-16-54i3	9630046K23Rik	RIKEN cDNA 9630046K23 gene	224143	Mm.284366	NM_172380	1.44	1.58	0.13	16 B3
MG-6-2i22	Timp2	tissue inhibitor of metalloproteinase 2	21858	Mm.206505	NM_011594	1.42	1.5	0.08	11 72.0 cM
MG-13-1c9	S100a10	Calpactin I light chain	20194	Mm.1	NM_009112	1.41	1.48	0.05	3 41.7 cM
MG-14-79j4	Lhfp12	Lipoma HMGIC fusion partner-like 2, mRNA	218454	Mm.316553	NM_172589	1.41	1.58	0.21	13 C3-D1
MG-3-38p22	Ctsl	Cathepsin L precursor	13039	Mm.930	NM_009984	1.38	1.63	0.15	13 30.0 cM
MG-3-91d9	Kcnh2	Potassium voltage-gated channel, sub family H (eagralated), member 2	16511	Mm.6539	AC113055	1.38	1.53	0.1	5
MG-3-45k24	Gsn	Gelsoin	227753	Mm.21109	NM_146120	1.37	1.48	0.15	2 24.5 cM
MG-6-31m16	Clec21	C-type lectin domain family, member 1	381758	Mm.349066	XM_355753	1.37	1.4	0	6 B1
MG-13-2e2	EST	EST			CR517795	1.36	1.48	0.1
MG-3-171n1	Iap1-3	Intracisternal A particles, Eya 1 linked	15601		AF097546	1.35	1.5	0.08	1 10.4 cM
MG-3-85d19	Sfrsi	Splicing factor, arginine/serine-rich (ASF/SFZ)		Mm.45645	CR518131	1.34	2.05	0.89	11
MG-12-195b2	Timp3	Metalloproteinase inhibitor 3 precursor (TIMP3) tissue inhibitor	21859	Mm.4871	NM_011595	1.33	1.45	0.13	10 47.0 cM
MG-14-58h24	Col8a1	Procollagen type VIII, α 1	12837	Mm.370175	BC058281	1.33	1.45	0.1	16 C1.1
MG-3-23b6	Tpm1	Tropomyosin 1 α	22003	Mm.121878	M22479	1.32	1.38	0.05	9 40.0 cM
MG-13-1k15	S100a4	S100 calcium binding protein A4	20198	Mm.3925	BC051214	1.31	1.45	0.13	3 43.6 cM
Table 3b. cont.	S100a1	S100 calcium binding protein A1	20193	Mm.24662	NM_011309	1.31	1.35	0.06	3 43.6 cM
MG-3-72e5
MG-68-98m4	Ctsz	Cathepsin Z precursor	64138	Mm.156919	NM_022325	1.31	1.4	0.08	2 103.5 cM
MG-13-117j9	Ahnak	AHNAK nucleoprotein (desmoyokin), mRNA	66395	Mm.203866	BC006892	1.29	1.4	0.08	19 A
MG-15-217f23	A930026I22Rik	RIKEN cDNA A930026I22 gene	77970	Mm.259790	BX632387	1.28	1.38	0.05	19 C3
MG-6-30n6	S100a6	S100 calcium binding protein A6 (Calcyclin)	20200	Mm.100144	NM_011313	1.28	1.45	0.1	3 43.6 cM
MG-6-30o19	Clstn3	calsyntenin 3	232370	Mm.193701	NM_153508	1.28	1.35	0.06	6 F2
MG-6-31b18	Camkk2	Calcium/calmodulin-dependent protein kinase kinase 2 β	207565	Mm.289237	NM_145358	1.28	1.3	0	5 F
MG-12-3f11	S100g	S100 calcium binding protein G	12309	Mm.6891	NM_009789	1.27	1.3	0	X F4
MG-6-1g19	My16	myosin, light polypeptide 6, alkali, smooth muscle and non-muscle	17904	Mm.337074	NM_010860	1.27	1.35	0.06	10
MG-6-31b24	4933433P14Rik	RIKEN cDNA 4933433P14 gene	66787	Mm.248019	BC031494	1.27	1.4	0.08	12 E
MG-12-42l4	Cotl1	Coactosin-like 1 (Dictyostelium)	72042	Mm.141741	XM_489250	1.26	1.45	0.13	8 E1
MG-3-254m20	A430065p19	Riken A430065P19 gene	329421	Mm.99648	NM_177376	1.26	1.45	0.13	2 C2
MG-3-37p2	Qscn6	Quiescin Q6	104009	Mm.27035	NM_001024945	1.26	1.38	0.05	1 G3
MG-6-31h10	Map3k11	mitogen activated protein kinase kinase kinase 11	26403	Mm.185020	NM-022012	1.26	1.33	0.05	19 0.5 cM
MG-6-82c15	Dbp	D site albumin promoter binding protein	13170	Mm.378235	NM_016974	1.26	1.3	0	7 23.0 cM
MG-8-17n2	Stard9	START domain containing 9	211824	Mm.246506	XM_619801	1.26	1.38	0.05	2 F1
MG-14-95h3	Ggta1	Glycoprotein galactosyltransferase α 1	14594	Mm.281124	NM_010283	1.25	1.38	0.15	2 25.0 cM
MG-3-6912	AC116589				AC116589	1.25	1.4	0.08	13

Table 4 Primer sequences and polymerase chain reaction conditions of genes selected for real time polymerase chain reaction assays

Gene symbol	Gene name	Primer sequence (5' to 3')	Product size (bp)	Annealing T°
S100a4	S100 calcium binding protein A4	For-TCAAGCTGAACAAGACAGAGC	131	56
		Rev-ACTTCATTGTCCCTGTTGCTG
S100a6	S100 calcium binding protein A6	For-TCCACAAGTACTCTGGCAAGG	138	56
		Rev-GGTCCAGATCATCCATCAGC
S100a10	S100 calcium binding protein A10	For-AGCTCTTCCAAGGACTGCTG	138	60
		Rev-TTTGTCAAGTGGTCTTTGTCG
Camkk2	Calcium/calmodulin-dependent protein kinase kinase2, β	For-GTCACACCACGTCTCCATTAC	144	56
		Rev-ACTTTCATTGCGTAATAAGTATTGTC
Tpm1	Tropomyosin 1 α	For-ATGCCCGTGTTCCTTAAAGC	157	59
		Rev-CCCTGACATGGAGAACTGGG
Sparc	Secreted acidic cystein rich glycoprotein	For-ATCCCCATGGAACATTGCAC	122	59
		Rev-TCCTTGTTGATGTCCTGCTCC
Trh	Thyrotropin releasing hormone	For-GGTTCTTCCACGCCTCCTAAG	135	59
		Rev-AACCTTGGAGGATGCGCTG
Lefty1	Left right determination factor 1	For-GCAAACCAAGGACAGAATCCC	108	59
		Rev-TGTTTGCCCAACTTGTGGC
P2rx1	Purinergic receptor P2X, ligand-gated ion channel 1	For-GTCTCCAGGCTTCAACTTC	156	56
		Rev-GAGCCGATGGTAGTCATAGT
P2rx2	Purinergic receptor P2X, ligand-gated ion channel 2	For-GTAGAGCAAGCAGGAGAGA	89	60
		Rev-AGACAAGTCCAGGTCACAG
P2rx3	Purinergic receptor P2X, ligand-gated ion channel 3	For-CCCGCTAAGACCTGAATCT	142	60
		Rev-AGTCCCTGGTATGTGGTAGG
P2rx4	Purinergic receptor P2X, ligand-gated ion channel 4	For-CTGTGTGACGTCATAGTCC	85	60
		Rev-GCTCGTAGTCTTCCACATAC
P2rx5	Purinergic receptor P2X, ligand-gated ion channel 5	For-GCTTTCTTCTGTGACCTG	99	60
		Rev-ATCTTCCTCCTTCTGACC
P2rx6	Purinergic receptor P2X, ligand-gated ion channel 6	For-CCCAAAGACGACTACCAA	86	60
		Rev-CAAACCACTCCTAGGACACT
P2rx7	Purinergic receptor P2X, ligand-gated ion channel 7	For-GGAAGTTAACCGTTCCTG	98	60
		Rev-TGGGCTAGACCTACTTCC
P2ry1	Purinergic receptor P2Y, G-protein coupled 1	For-GGTCTAGCAAGTCTCAACAG	121	60
		Rev-GTAAATTGGCCTCACTCC
P2ry2	Purinergic receptor P2Y, G-protein coupled 2	For-CCAAGCATGGAGAGGAGT	156	60
		Rev-GAATTGCTTGCACCACAG
P2ry4	Purinergic receptor P2Y, G-protein coupled 4	For-ACTAACTGCAGGCAGAGG	170	60
		Rev-CCAGCAAAGAGTACTGAGG
P2ry5	Purinergic receptor P2Y, G-protein coupled 5	For-CATGTACCCGATCACTCTC	136	60
		Rev-GAACCTGGAGTCACTTCTTC
P2ry6	Purinergic receptor P2Y, G-protein coupled 6	For-GAGTTCTGCGTGTGTGTG	172	60
		Rev-GTCAGCCTTTCCTATGCT
P2ry10	Purinergic receptor P2Y, G-protein coupled 10	For-GACATTTGGTATGCAGGCAAG	82	56
		Rev-TGGTCCCTTCCTCTTCCTTAGT
P2ry12	Purinergic receptor P2Y, G-protein coupled 12	For-TGCTGAGGTGCTCAAACTCTAC	84	56
		Rev-GGGTCTCTTCGCTTGGTTC
P2ry13	Purinergic receptor P2Y, G-protein coupled 13	For-AACAGAGCACCAGAAGAGAG	119	60
		Rev-AGGATGCAGATGCTGTTG
P2ry14	Purinergic receptor P2Y, G-protein coupled 14	For-CACAAAGAGTCAGACGGAAGG	96	60
		Rev-ACATTGGCAGCCGAGAGTAG
Bdkrb1	Bradykinin receptor, β 1	For-AGTCGTCCCTGATCTGAA	85	56
		Rev-GTTCAACTCCACCATCCT
Bdkrb2	Bradykinin receptor, β 2	For-CACGCAGATCAGTTCCTAC	99	60
		Rev-ACCTCTCGGGACTTCTTC
Hrh1	Histamine receptor H1	For-GGGCTACATCAACTCCAC	153	60
		Rev-CCCTCTTGGACATCAGAC
Hrh2	Histamine receptor H2	For-CCTCTCCTTCCTCTCTATTC	110	60
		Rev-CCACCAGTCCATATACCTC
Hrh3	Histamine receptor H3	For-ACAGTCAGCAGGAGGGAGAG	135	56
		Rev-TGTCTTCACATTGGCAGAGG
Hrh4	Histamine receptor H4	For-CTTGGAAGAACAGCACGAAC	106	56
		Rev-GAGATGACAGGAAGCAGGAA
Ryr1	Ryanodine receptor 1, skeletal muscle	For-ACTTTGTACCCTGTCCTGTG	132	60
		Rev-CATAGGTCCATCCTTGCTC
Ryr2	Ryanodine receptor 2, cardiac muscle	For-GGATGAATGTCTCACTGTCC	147	60
		Rev-CTTATGTGGCTTCCACTCC
Ryr3	Ryanodine receptor 3	For-CAGGTATCTTGGAGGTCTTG	111	60
		Rev-GCCCATGCTTATCCAGTAG
Itpr1	Inositol 1,4,5-triphosphate receptor 1	For-GGTTGATGACCGTTGTGTTG	121	60
		Rev-GAACTGTTTCTGTGCGGAGT
Itpr2	Inositol 1,4,5-triphosphate receptor 2	For-GTGAGGATGGCATAGAAAG	165	60
		Rev-AGAAGAACAGGAGGTCGTAG
Itpr3	Inositol 1,4,5-triphosphate receptor 3	For-GGCTTCATCAGCACTTTGG	96	60
		Rev-GCAATCTCGGAACTTCTTGG
H2afz	H2A histone family, member Z	For-ACAGCGCAGCCATCCTGGAGTA	202	60
		Rev-TTCCCGATCAGCGATTTGTGGA
Ppia	Peptidylprolyl isomerase A	For-CGCGTCTCCTTCGAGCTGTTTG	150	60
		Rev-TGTAAAGTCACCACCCTGGCACAT