|Chimera Development Via ES Cell Injection|
Gene targeting is carried out in mouse embryonic stem (ES) cells in vitro. Successfully targeted ES cells are then injected into the blastocoel of 3.5 day old mouse blastocysts. Injected embryos are transferred surgically to the uterine horns of appropriately timed pseudopregnant recipient females which are allowed to gestate normally. If the targeted ES cell clone contributes to the development of the fetus, the pups produced will be chimeras, exhibiting patches of coat color from the host embryo and patches from the injected ES clone. Chimeric mice must be crossed with wildtype mice to determine if the ES cells can “go germline,” meaning they can contribute to the germline of the chimera and produce offspring arising from an ES cell-derived gamete.
Identification Of Chimeras
Chimeras are usually identified by coat color. ES cells derived from black mice are injected into fertilized embryos from white mice. And ES cells derived from agouti mice are typically injected into fertilized embryos from black mice. Generally, the higher the coat color contribution from the ES cell line, the more likely it is that the mutation will go germline. Targeted G4 and R1 ES cells (agouti) are injected into C57BL/6 embryos (black), and chimeras are identified as agouti on black coat coloration. Targeted C57BL/6 ES cells (black) are injected into albino (tyrosine deficient) C57BL/6 embryos (white with pink eyes), and chimeras are identified as black on white coat coloration. Coat color pattern and percentage can be determined approximately 7 days after birth, and sex determined by 3 weeks of age. Chimeric pups are typically weaned at 3–4 weeks of age.
The contribution to the germline by the ES cell lineage in any given chimera is unknown. On average, lower-percentage chimeras (those with less ES cell derived fur) are less likely to have ES cell-derived gametes. Nevertheless, a high degree of chimerism is not a guarantee of germline transmission. Therefore, it is generally prudent to breed all of the chimeric males (>30% ES coat color contribution). It may be necessary to produce several litters of pups before the first coat-color germline pup is obtained. Since most targeted ES cells are heterozygous, expect about half of the coat-color germline pups to test positive.
Although chimeras can be either males or females, generally the males are the only ones that will go germline, if they are made with XY ES cells. Since the vast majority of ES cell lines are XY, it is considered a good sign when a majority of the chimeras are males. Strong contributions by ES cells to the germline can cause a female blastocyst to develop into a phenotypically male chimera. Female mice occasionally carry the targeted mutation in the germline, but only if the ES line has lost the Y chromosome, making it XO. If the sex ratio of the resulting litter is not skewed toward males, it is advisable to mate strongly chimeric females.
A method to determine the wildtype allele from the mutant allele must be developed to distinguish the 3 possible genotypes arising from heterozygous matings. As an additional measure, tissue from the heterozygotes should be analyzed by Southern Blot to confirm the targeted mutation has remained intact from the culture dish through to germline transmission.
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Reserve An Injection Session
To order an ES cell injection session, go to our TRP Forms page. Fill out a “Request for Targeted ES Cell Injection for Blastocysts” form and e-mail it to Bob Hunter firstname.lastname@example.org. Be sure to include a current budget number and your IACUC animal use protocol number.