title: Gene targeting and Calcium handling efficiencies in mouse embryonic stem cell lines.
abstract: OBJECTIVE
  To compare gene targeting efficiencies, expression profiles, and Ca(2+) handling potentials in two widely used mouse embryonic stem cell lines.
  
  
  METHODS
  The two widely used mouse embryonic stem cell lines, R1 and HM-1, were cultured and maintained on Mitomycin C treated mouse embryonic fibroblast feeder cell layers, following standard culture procedures. Cells were incubated with primary and secondary antibodies before fluorescence activated cell sorting analysis to compare known pluripotency markers. Moreover, cells were harvested by trypsinization and transfected with a kinase-inactive murine Tyk2 targeting construct, following the BioRad and Amaxa transfection procedures. Subsequently, the cells were cultured and neomycin-resistant cells were picked after 13 d of selection. Surviving clones were screened twice by polymerase chain reaction (PCR) and finally confirmed by Southern blot analysis before comparison. Global gene expression profiles of more than 20 400 probes were also compared and significantly regulated genes were confirmed by real time PCR analysis. Calcium handling potentials of these cell lines were also compared using various agonists.
  
  
  RESULTS
  We found significant differences in transfection efficiencies of the two cell lines (91% ± 6.1% vs 75% ± 4.2%, P = 0.01). Differences in the targeting efficiencies were also significant whether the Amaxa or BioRad platforms were used for comparison. We did not observe significant differences in the levels of many known pluripotency markers. However, our genome-wide expression analysis using more than 20 400 spotted cDNA arrays identified 55 differentially regulated transcripts (P < 0.05) implicated in various important biological processes, including binding molecular functions (particularly Ca(2+) binding roles). Subsequently, we measured Ca(2+) signals in these cell lines in response to various calcium agonists, both in high and low Ca(2+) solutions, and found significant differences (P < 0.05) in the regulation of Ca(2+) homeostasis between the investigated cell lines. Then we further compared the detection and expression of various membrane and intracellular Ca(2+) receptors and similarly found significant (P < 0.05) variations in a number of calcium receptors between these cell lines.
  
  
  CONCLUSIONS
  Results of this study emphasize the importance of considering intrinsic cellular variations, during selection of cell lines for experiments and interpretations of experimental results.
authors: Solomon Mamo, Julianna Kobolak, Andras Dinnyes, Genetic Reprogramming Group, Agricultural Biotechnology Center, Szent-Gyorgyi Albert ut. 4, H-2100 Gödöllő, Hungary.
authors: Solomon
authors: S
authors: Mamo
authors: Julianna
authors: J
authors: Kobolak
authors: Istvan
authors: I
authors: Borbíró
authors: Tamás
authors: T
authors: Bíró
authors: Istvan
authors: I
authors: Bock
authors: Andras
authors: A
authors: Dinnyes
references: Nucleic Acids Res. 1996 Feb 15;24(4):596-601
references: 8604299
references: Dev Biol. 2004 May 15;269(2):360-80
references: 15110706
references: Blood. 1998 Feb 15;91(4):1332-40
references: 9454764
references: J Allergy Clin Immunol. 2007 Aug;120(2):300-7
references: 17507084
references: Mamm Genome. 2001 Oct;12(10):772-8
references: 11668392
references: Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8424-8
references: 8378314
references: Genome Biol. 2003;4(5):P3
references: 12734009
references: Genome Biol. 2003;4(10):R70
references: 14519205
references: Nature. 1993 Jan 28;361(6410):315-25
references: 8381210
references: Proc Natl Acad Sci U S A. 1981 Dec;78(12):7634-8
references: 6950406
references: Endocr Rev. 1993 Feb;14(1):40-58
references: 8491154
references: Nat Biotechnol. 2008 Mar;26(3):313-5
references: 18278034
references: Dev Biol. 2004 Aug 15;272(2):483-96
references: 15282163
references: BMC Cell Biol. 2009 Jun 05;10:44
references: 19500347
references: Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5128-32
references: 1594621
references: J Mol Biol. 2005 Jun 10;349(3):487-500
references: 15896347
references: Mol Cell Biol. 1992 Aug;12(8):3365-71
references: 1321331
references: Biochem J. 1984 Mar 1;218(2):475-82
references: 6324757
references: Neurosci Lett. 2003 Jan 9;336(1):9-12
references: 12493590
references: Nature. 1981 Jul 9;292(5819):154-6
references: 7242681
references: Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1961-6
references: 9050887
references: Nature. 1993 Sep 2;365(6441):87-9
references: 8361547
references: ScientificWorldJournal. 2006 Oct 09;6:1247-61
references: 17041716
references: Cell Signal. 2001 Aug;13(8):565-74
references: 11483409
references: Nucleic Acids Res. 1992 Jul 25;20(14):3795-6
references: 1641353
references: Somat Cell Mol Genet. 1992 Jul;18(4):325-36
references: 1440055
references: Neuroscience. 2002;110(2):329-38
references: 11958874
references: PLoS Biol. 2003 Dec;1(3):E74
references: 14691545
references: BMC Cell Biol. 2008 Dec 18;9:69
references: 19094237
references: Nucleic Acids Res. 2003 Jan 15;31(2):E1-1
references: 12527790
references: Nature. 1998 Apr 30;392(6679):863, 865-6
references: 9582065
references: Agents Actions. 1990 Jan;29(1-2):8-15
references: 2139301
references: Nat Biotechnol. 2000 Apr;18(4):399-404
references: 10748519
references: Nat Biotechnol. 2000 Jan;18(1):33-7
references: 10625387
references: Stem Cells. 2005 Feb;23(2):166-85
references: 15671141
references: Am J Reprod Immunol. 2006 Mar;55(3):169-80
references: 16451351
references: Nat Rev Mol Cell Biol. 2000 Oct;1(1):11-21
references: 11413485
references: Nat Rev Genet. 2006 Mar;7(3):200-10
references: 16485019
references: BMC Dev Biol. 2007 Mar 06;7:14
references: 17341302
references: J Physiol. 1996 Nov 1;496 ( Pt 3):617-25
references: 8930830
references: Exp Cell Res. 2004 Feb 15;293(2):229-38
references: 14729460
references: BMC Cell Biol. 2008 Jul 11;9:37
references: 18620564
references: PLoS One. 2010 Feb 09;5(2):e9121
references: 20161753
references: Physiol Rev. 2001 Apr;81(2):767-806
references: 11274344
references: Annu Rev Physiol. 1994;56:463-84
references: 8010748
references: Stem Cells. 2004;22(4):531-43
references: 15277699
references: Stem Cells. 2006 Feb;24(2):454-61
references: 16099993
references: Exp Dermatol. 2008 May;17(5):405-11
references: 18312380
references: Cell Calcium. 2004 Aug;36(2):135-46
references: 15193861
references: Clin Allergy Immunol. 2002;17:27-64
references: 12113220