Abstract
Genetic modification of mouse embryonic stem cells is a powerful method to study gene function in whole animal models. The ability to re-design genes in mouse to reproduce genetic defects found in human patients gives researchers a wide open arena for biomedical research. Successful manipulation of ES cells requires culture conditions that restrain differentiation and support robust growth. It is essential to use culture conditions that are carefully calibrated to maintain ES cells in pluripotent condition because the only test for germline transmission is the time-consuming process of preparing ES cell-mouse chimeras and breeding them for several months. Fastidious adherence to culture conditions will maintain the capability of ES cells to differentiate into many cell types. Inadequate care of ES cells will degrade the ES cell quality and result in cells that fail to transmit mutant genes through the germline. Mouse ES cell culture is a well-established procedure. After targeting vector electroporation and identification of euploid gene-targeted clones, generation of chimeras from three clones is normally sufficient to transmit engineered mutations through the germline and produce a new mouse strain. Although the process of generating a new mouse model is not always routine, careful attention to ES cell culture is rewarded by the production of robust gene-targeted ES cell clones that regularly go germline.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Abbreviations
- 2-ME:
-
Beta-Mercaptoethanol
- DMEM:
-
Dulbecco’s Modified Eagle Medium
- DMSO:
-
Dimethyl sulfoxide
- D-PBS:
-
Dulbecco’s phosphate buffered saline
- ES cells:
-
Embryonic stem cells
- FBS :
-
Fetal bovine serum
- FIAU:
-
1-(-2-Deoxy-2-fluoro-1-b-d-arabino-furanosyl)-5-iodouracil
- LIF:
-
Leukemia Inhibitory Factor (ESGRO ®)
- MEF :
-
Mouse embryonic fibroblast feeder cells
- MEM:
-
Minimal Essential Medium
- Pen/strep:
-
Penicillin/streptomycin antibiotic mix for cell culture
- SDS:
-
Sodium dodecyl sulfate
References
Kim I, Saunders TL, Morrison SJ (2007) Sox17 dependence distinguishes the transcriptional regulation of fetal from adult hematopoietic stem cells. Cell 130:470–483
Lapinski PE, Bauler TJ, Brown EJ, Hughes ED, Saunders TL, King PD (2007) Generation of mice with a conditional allele of the p120 Ras GTPase-activating protein. Genesis 45:762–767
Huang X, Fu Y, Charbeneau RA, Saunders TL, Taylor DK, Hankenson KD, Russell MW, D'Alecy LG, Neubig RR (2006) Pleiotropic phenotype of a genomic knock-in of an RGS-insensitive G184S Gnai2 allele. Mol Cell Biol 26:6870–6879
Wada M, Saunders TL, Morrow J, Milne GL, Walker KP, Dey SK, Brock TG, Opp MR, Aronoff DM, Smith WL (2009) Two pathways for cyclooxygenase-2 protein degradation in vivo. J Biol Chem 284:30742–30753
Capecchi MR (1994) Targeted gene replacement. Sci Am 270:52–59
Hughes ED, Qu YY, Genik SJ, Lyons RH, Pacheco CD, Lieberman AP, Samuelson LC, Nasonkin IO, Camper SA, Van Keuren ML, Saunders TL (2007) Genetic variation in C57BL/6 ES cell lines and genetic instability in the Bruce4 C57BL/6 ES cell line. Mamm Genome 18:549–558
Liu X, Wu H, Loring J, Hormuzdi S, Disteche CM, Bornstein P, Jaenisch R (1997) Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission. Dev Dyn 209:85–91
Sugawara A, Goto K, Sotomaru Y, Sofuni T, Ito T (2006) Current status of chromosomal abnormalities in mouse embryonic stem cell lines used in Japan. Comp Med 56:31–34
Tucker KL, Wang Y, Dausman J, Jaenisch R (1997) A transgenic mouse strain expressing four drug-selectable marker genes. Nucleic Acids Res 25:3745–3746
Linnell ER, Lerner CP, Johnson KA, Leach CA, Ulrich TR, Rafferty WC, Simpson EM (2001) Transgenic mice for the preparation of puromycin-resistant primary embryonic fibroblast feederlayers for embryonic stem cell selection. Mamm Genome 12:169–171
O'Connell RC, Wittler RG, Faber JE (1964) Aerosols as a source of widespread mycoplasma contamination of tissue cultures. Appl Microbiol 12:337–342
Longo L, Bygrave A, Grosveld FG, Pandolfi PP (1997) The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism. Transgenic Res 6:321–328
Kumar RA, Chan KL, Wong AH, Little KQ, Rajcan-Separovic E, Abrahams BS, Simpson EM (2004) Unexpected embryonic stem (ES) cell mutations represent a concern in gene targeting: lessons from “fierce” mice. Genesis 38:51–57
Martin GR, Evans MJ (1975) Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro. Proc Natl Acad Sci USA 72:1441–1445
Chen YT, Bradley A (2000) A new positive/negative selectable marker, puDeltatk, for use in embryonic stem cells. Genesis 28:31–35
Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC (1993) Derivation of completely cell culture-derived mice from early passage embryonic stem cells. Proc Natl Acad Sci USA 90:8424–8428
Tybulewicz VLJ, Crawford CE, Jackson PK, Bronson PT, Mulligan RC (1991) Neonatal lethality and lymphopenia in Mice with a homozygous disruption of the c-abl proto-oncogene. Cell 65:1153–1163
Yagi T, Nada S, Watanabe N, Tamemoto H, Kohmura N, Ikawa Y, Aizawa S (1993) A novel negative selection for homologous recombinants using diphtheria toxin A fragment gene. Anal Biochem 214:77–86
Schaft J, Ashery-Padan R, van der Hoeven F, Gruss P, Stewart AF (2001) Efficient FLP recombination in mouse ES cells and oocytes. Genesis 31:6–10
Yu H, Kessler J, Shen J (2000) Heterogeneous populations of ES cells in the generation of a floxed Presenilin-1 allele. Genesis 26:5–8
Yu RN, Ito M, Saunders TL, Camper SA, Jameson JL (1998) Role of Ahch in gonadal development and gametogenesis. Nat Genet 20:353–357
Nagy A, Gertsenstein M, Vintersten K, Behringer R (2003) Manipulating the mouse embryo: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Ramirez-Solis R, Rivera-Perez J, Wallace JD, Wims M, Zheng H, Bradley A (1992) Genomic DNA microextraction: a method to screen numerous samples. Anal Biochem 201:331–335
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer-Verlag Berlin Heidelberg
About this protocol
Cite this protocol
Hughes, E.D., Saunders, T.L. (2011). Gene Targeting in Embryonic Stem Cells. In: Pease, S., Saunders, T. (eds) Advanced Protocols for Animal Transgenesis. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-20792-1_14
Download citation
DOI: https://doi.org/10.1007/978-3-642-20792-1_14
Published:
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-20791-4
Online ISBN: 978-3-642-20792-1
eBook Packages: Springer Protocols