Blastocyst Injection Service

There are two methods to produce chimeric mice from mouse embryonic stem cells (ESCs). The traditional method is the microinjection of 3.5 dpc blastocysts with gee targeted ESCs. This nearly always produces what are call chimeras. While these are truely chimeric mice (derived from the host blastocyst and the ESC graft, they are nearly always scored by coat color chimerism. While coat color chimerism does correlate with germ line transmission of the grafted ESCs, it is not a direct correlation. That is, 100% coat color chimeras often do not transfer the gene. Germ line transmission is customarily determined by mating the male chimeras and not the female chimeras. This is for two reasons, A. the ES cells are male cells that cause sex conversion when the  ES cells contribute to the gonadal primordial cells and B. males can produce many more mice in a shorter time when harem bred. Traditionally, ESCs that have a dominant coat color gene that contrasts with the host blastocyst are used. Hisorically, this was the 129 strain ESCs (which contained a dominant agouti coat color allele like brown field mice) and C57Bl/6 host embryos.  This combination produced "brown" and black coat color chimeras. When these male chimeras were bred to C57Bl/6  females any agouti colored pups were ESC-derived. This made for the easy identification of germ line chimeras. It must be noted that the segregation of alleles that occurs during meiosis nearly always means that only half of the agouti pups will be founder that carry the gene-targeted allele.

With the growing availability of C57Bl/6 (B6) ESCs from the KOMP and IGTC the vast majority of gene-targetings have switched to this cell line which has been a long time of the preference of immunologists. B6 ESCs are generally recognized to be more demanding of cell culture conditions and have a reduced propensity to make high percentage coat color chimeras.  These are usually produced by injecting the B6 ESCs which are from a homozygous black mouse into an albino host blastocyst. The most commonly used host blastocysts are from Balb/c mice and  albino B6 mice that are homozygous for a mutation it the pigment essential TryC, or tyrosinase C gene. There are relatively few early passage B6 ESC lines available. This can make the derivation of germ line chimeras more difficult form blastocyst injection as has been widely reported. However, there is a complementary method to produce germ lime chimeras from B6 ESCs. This method pioneered by Andras Nagy in Toronto is call "embryo aggregation". With B6 ESCs, the Core will do diploid embryo aggregation in parallel with blastocyst injection. This method involved the cocultivation of ESCs with 2.5 dpc embryos to produce blastocysts which are then transferred into pseudo pregnant females. There is some evidence that this method nicely complements blastocyst injections especially with B6 ESCs. B6 chimeras from either method should be bred to albino B6 females and colored pups are germ line derived.

At this point the Core does not have enough space in the MTF barrier vivarium to breed the chimeras.  Therefore the SPF chimeras will be turned over to the requesting PI. The Core is glad to provide breeding recommendations to interrogate the germ line of  the chimeras delivered to the investigator. These recommendations will yield germ line transmission in the shortest period of time which is essential for grant funded projects.