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Mission
The Murine Embryonic Stem Cell Core has been created to help you create mutations in murine embryonic stem cells. The core has several missions, including development of state-of-the-art reagents for the production of targeted mutations in embryonic stem cells, the creation of quality-controlled embryonic stem cell lines, and the teaching of methods for embryonic stem cell culture and manipulation. The core provides quality-controlled cells developed here at Washington University. In addition, our core can provide a teaching service, so that "hands-on" production of targeted ES clones can be learned. Finally, the core will attempt to assist all users with blastocyst injection, if injection services are not available to that laboratory. By providing a comprehensive service, we hope to facilitate the production of gain-of-function and loss-of-function mouse models for our faculty.
- An available service /protocol for chimeric detection and quantification of B6/GFP cells (12/05/07)
- Cellular growth conditions under hypoxia: A Report (11/29/07)
- The ES Core recommends the following article:
E. Seong, et al, "To Knockout in 129 or in C57BL/J: That is the Question", TRENDS in Genetics, Vol. 20, No.2, February 2004, pp. 59-62
- The ES Core announces the release and availability of a new 129Sv/J ES line, named 'SCC#10', and derived from 129Sv/J (129X1Sv/J in the newer nomenclature) mice from The Jackson Laboratories. This line has a normal, 40, XY karyotype, has been MAP tested, is free of mycoplasma, and has produced chimeras that have given germ-line transmission.
- The ES core now has R1 ES cells available for transfections within the Washington University Medical School community. Please note that this is a hybrid line (129/Sv X 129/Sv-CP F1). These cells were obtained from Dr. Andras Nagy. Details about the R1 ES line can be found at Dr. Nagy's webpage:
- The ES Core announces the release of a new ES cell line, B6/Blu-1
- The ES Core announces the release and availability of a new 129SvEv ES line, named 'EDJ 22', and derived from 129SvEv (129S6SvEv in the newer nomenclature) mice from Taconic. This line has a normal, 40, XY karyotype, has been MAP tested, is free of mycoplasma, and has produced chimeras that have given germ-line transmission.
- The ES Core announces the release of a new ES cell line, B6/GFP (C57BL/6-Tg(Actb-EGFP)OsbY01)
Before beginning gene targeting experiments, please be sure to develop a good genomic map of the targeted locus and unique probes that are external to your targeting construct. After extraction of the DNA from your ES clones, you will have only enough DNA for a single restriction digest and Southern analysis. Therfore, it is imperative that your methodology for detecting recombinant alleles has been worked out prior to the screening of your clones. This facility cannot provide the genomic DNA needed for you to develop a map of your locus. We recommend that you isolate genomic 129X1/SvJ DNA from the tails or organs of 129X1/SvJ mice (Jackson Laboratories, Cat.# 000691). A protocol for producing high quality tail DNA can be obtained in the protocols section within this site.
To achieve optimal efficiency of homologous recombination, it may be important for your targeting arms to be made from DNA that is isogenic for the embryonic stem cell line that you are using. In our core, we use SCC#10 embryonic stem cells, which are derived from 129X1/SvJ mice. 129X1/SvJ mice have recently been found to be "contaminated" by another unknown strain of mouse (see Threadgill et al, Mammalian Genome 8: 390-393, 1997, and also Simpson et al, Nature Genetics 16: 19-27, 1997). "Contamination" means that there appear to be parts of the 129X1/SvJ genome that are not derived from 129 mice; apparently, this 129 line was outbred at some time in the distant past, and these DNA sequences are the genetic remnants of that event. It is important to note that there is no evidence that SSC#10 embryonic stem cells have drifted from the 129X1/SvJ background; all polymorphic markers examined are identical to that of 129X1/SvJ mice (see Simpson et al, Nature Genetics). If you make your targeting arms from 129 DNA of another source, it is possible that your DNA may not be isogenic for 129X1/SvJ mice (if your gene lies in one of the "non-129" regions). Threadgill's paper contains a very nice map of the regions that are not clearly from 129 DNA, and this map should be consulted as you plan your targeting construct. Isogenic DNA is not always required for efficient targeting, but it is the safest technique, if you have a choice.
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Murine Embryonic Stem Cell Core The Alvin J. Siteman Cancer Center at Washington University St. Louis, Missouri 63110
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