ES Cell Microinjection
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.
- purchase and housing of mice used to produce embryos
- thaw and expand investigator’s targeted ES cell clone for injection
- superovulation of embryo donors and mating with stud males
- creation of pseudopregnant foster mothers by mating outbred ICR females with vasectomized males and checking for vaginal plugs the morning of injection
- harvesting of embryos from euthanized donors
- injection of expanded blastocysts as possible with ~10-12 ES cells each
- surgical transfer of injected blastocysts into the uterine horns of E2.5 dpc pseuodpregnant mothers
- monitoring of implanted/pregnant females before and after birth of pups
- identification of chimeric pups by coat color and determination of sex
- weaning up pups at day 21
- arrange transfer of chimeras to the investigator’s mouse colony
- germline testing of chimeras to assess germline transmission is available for an additional fee
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 and typically injected into fertilized embryos from black or white 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/6J embryos (white with pink eyes), and chimeras are identified as black on white coat coloration. Coat color pattern and percentage can be determined approximately 10 days after birth, and sex determined by 2 weeks of age. Chimeric pups are typically weaned at 3 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.
- G4 chimeras are mated to C57BL/6 to test for germline transmission. Germline offspring can be agouti OR black because the G4 is an F1 hybrid of C57BL/6 x 129. The black pups should all be genotyped.
- R1 (129) chimeras are mated to C57BL/6 to test for germline transmission. Germline offspring are identified by an agouti coat color. Black pups are derived from the wild type C57BL/6 host embryos.
- C57BL/6 ES cell chimeras should be bred back to albino C57BL/6 females. Black pups are germline and white pups are wild type.
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.
- We guarantee that at least 24 injected embryos will be implanted into pseudopregnant recipients, or 2 chimeric pups will be produced, whichever comes first.
- C57BL/6 ES cells are significantly less efficient compared to hybrid G4 ES cells, therefore it is often necessary to inject 2 or 3 different targeted clones in order to produce chimeras from C57BL/6 ES cells.
- We will, after consultation, inject targeted ES cells provided from outside vendors (KOMP, EUCOMM, etc.) but we cannot guarantee that these clones will produce chimeras, or that chimeras will go germline. Overall, we have found the success rate (as defined by production of chimeras) very low for the consortium clones.
- Cancellation of injection within two weeks of the scheduled date will result in a $500 fee to cover the cost of mice, hormones, media, and technician time.
- Click here for current rates.