Introduction

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. 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. On average, half of such offspring should inherit the targeted gene.

Service Description

• purchase and housing of mice used to produce embryos
• thaw and expand investigator’s targeted ES cell clone for injection
• superovulation of egg donors and mating with stud males
• creation of pseudopregnant foster mothers by mating outbred ICR females with vasectomized males
• harvesting of eggs from euthanized donors
• injection of as many expanded blastocysts as possible with about 12-15 ES cells each
• surgically transfer injected blastocysts to the uterine horns of 2.5 dpc foster mothers, or to the oviducts of 0.5 dpc foster mothers
• monitoring of foster mothers before and after birth of pups
• identification of chimeric pups by coat color and determination of sex
• arrange and assist with the transfer or shipment of chimeric mice to the investigator’s mouse colony

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 4 weeks of age.

Breeding Chimeras

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. Using this technique, wild type offspring can be distinguished at birth by the presence of pink eyes.

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.

Prices and Guarantees

• We guarantee that at least 40 injected embryos will be implanted into pseudo pregnant recipients, or 3 chimeric pups will be produced, whichever comes first.
• C57BL/6 ES cells are injected into albino C57BL/6 embryos. An extra $250 will be charged for the additional cost of purchasing albino C57BL/6 females.
• The investigator pays per diem cage charges beginning on the day of embryo transfer.
• Cancellation of injection within two weeks of the scheduled date will result in a $250 fee to cover the cost of the embryo donor mice purchased for the injection.
• Click here for current rates.

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 bhunter@uw.edu. Be sure to include a current budget number and your IACUC animal use protocol number.

Useful Links

International Society of Transgenic Technologies