ES Cell Injection Services


Injection of embryonic stem cells into mouse blastocysts results in chimeric mice, in which some cells in each tissue are derived from the ES cell lineage.  If the ES cells have been genetically modified, e.g., via gene targeting, those modifications can be passed on to offspring from the chimeras.  Since ES cells are usually heterozygous for the genetic modification, roughly half of the offspring arising from the ES cell lineage will carry the modification.  (See below for more details about breeding chimeras.)Similar to DNA microinjection to produce transgenic mice, our ES cell injection service involves the superovulation or hyperovulation and mating of blastocyst donors to obtain large numbers of embryos, and injected embryos are implanted into pseudopregnant foster mothers.

The client must supply at least one frozen vial of each ES cell clone to be injected.  Each clone must be tested for mycoplasma and mouse pathogens.  If this has not been done already, the TMF will require an extra vial of each clone for testing purposes.

Clients have the option of providing freshly-harvested cells on the day of injection, and we will discount the service fee by 10% if this is done.  However, this must be discussed with us well in advance of the injection, and evidence of pathogen testing with a satisfactory outcome will be required.

Several days before the injection, the TMF will thaw a vial of each clone to be injected, expand the cells, and pass them once.  On the morning of injection, the cells will be trypsinized, pelleted, resuspended in a small volume of medium and kept on ice until the actual injection.  If feeder cells are present, the trypsinized cell suspension will be plated onto a new well and incubated for about an hour to allow the feeder cells to attach to the plate.  Unattached ES cells are then removed with the medium, pelleted, and resuspended as before.

We can expand a clone and freeze back multiple tubes of cells for an additional fee.  If the client supplies a single vial for injection and does not ask us to expand it and freeze back extra vials, we will not be able to return any cells to the client after the injection.

We strongly encourage clients to have the average chromosome number determined for each clone.  In our experience, any clone in which less than 50% of the cells are euploid is probably not worth injecting.  Aneuploid cells can result in chimeras, but their genetic material rarely transmits through the germline.


Contact the TMF for a Service Request Form. The completed form must be signed by the Principal Investigator. Signed forms should be e-mailed to the TMF as a scanned image.

In addition to the Service Request Form, you must submit the following:

  1. Evidence that the ES cells are free of mouse pathogens (or ask us to test them);
  2. Evidence of approval from your institution’s Animal Care and Use Committee for your animal use protocol;
  3. For off-campus clients, a Material Transfer Agreement, signed in duplicate.

We also need the following information for each vial of cells:

  1. Genetic background of the ES cells (what mouse strain were they derived from).
  2. Approximate number of cells per vial.
  3. Type of medium in which the cells were cultured before freezing.
  4. Percentage of euploid cells.

Turnaround Times

The minimum time required to produce chimeric mice that can be transferred to the client is about 8 weeks, once we have ES cells ready for injection and the necessary paperwork. This includes about 2 weeks to order the blastocyst donors, 3 weeks gestation time, and 3 weeks from birth to weaning.

The turnaround time can be much longer, either because of previously scheduled orders or because of a failure to produce chimeras from the first injection.  However, if no chimeras are produced the first time and the total pup yield is reasonably normal, repeating the injection with the same clone is unlikely to be successful.

Performance Guarantees

For each day of injection, the TMF guarantees to inject at least 40 blastocysts and successfully transfer them to pseudopregnant foster mothers.  The client has the option of supplying one or two clones for each day of injection.  If two clones are supplied, roughly half of the blastocysts will be injected with each clone, but the guarantee of at least 40 injected blasts applies to the whole day of injection, not to each individual clone.

Service Description

The ES cell injection service includes:

  • Production or purchase of superovulated or hyperovulated and mated blastocyst donors.
  • Generation of pseudopregnant foster mothers by mating outbred ICR females with vasectomized males and checking for vaginal plugs the next day.
  • Injection of as many expanded blastocysts as possible with between 12-15 ES cells each.
  • Transfer of injected blastocysts to the uterine horns of 2.5 dpc foster mothers, or to the oviducts of 0.5 dpc foster mothers (dpc = days post-coitus).
  • Monitoring of foster mothers before and after birth of pups.
  • identification of chimeric pups by coat color and determination of sex (in cases where chimeras cannot be identified by coat color, the TMF will genotype offspring by PCR for a neo cassette or other suitable marker).
  • Transfer of chimeric pups to client after weaning.

If included on the order, we will also expand the ES cells and freeze back multiple vials.

ES cells from outside sources may need to be tested for mycoplasma and mouse pathogens at additional cost to the client.

Identification of Chimeras

Chimeras are usually identified on the basis of coat color (but see below for exceptions). Some ES cell lines are derived from one of the various 129 mouse lines, which have agouti (brown) fur. Blastocyst donors, by contrast, are usually C57BL/6 mice, which have black coats. Thus, chimeras from these lines have a mixture of black and agouti fur, with the agouti patches arising from the ES cell lineage.

Chimeras are often identified as being “strong” or “weak”, depending on how much of their fur is agouti, with stronger chimeras having more agouti fur. The strength of chimerism can also be expressed as a percentage of agouti fur, although this is a relatively inaccurate number. Chimeras with a large amount of ES cell-derived tissue are generally more likely to go germline, so it is considered a good sign when strong chimeras are produced from ES cell injection. However, we do not guarantee that any given chimera will go germline, since this is highly dependent on how the ES cells are handled during the gene targeting process.

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 (>85%) are males. Strong contributions by ES cells to the germline can cause a female blastocyst to develop into a chimera that is phenotypically male.  (By the same token, not all chimeras that appear to be male are capable of breeding, due to insufficient ES cell contribution to the germline of pups arising from female blastocysts.)

For the purpose of our performance guarantee, chimeras consist of any mice with visible agouti fur or carrying ES cell-specific markers, regardless of sex.

Coat color cannot be used to identify chimeras when an ES cell line derived from C57BL/6 mice is used, because these are also injected into C57BL/6 blastocysts.  One such line, JM8.N4, is currently available for gene targeting in our facility and we have verified that this cell line can contribute to the germline of mice following injection into blastocyst stage embryos. This line has been used for many of the ES cell clones being distributed by KOMP, EUCOMM, and other groups. It is derived from the C57BL/6NTac substrain. If such cells are injected into C57BL/6 blastocysts, all offspring will be completely black, regardless of the degree of chimerism. In this case, the offspring will be genotyped by the TMF, at no additional cost, to identify chimeras.

C57BL/6 ES cell lines can be injected into blastocysts from albino strains to allow identification of chimeras by coat color. However, most albino strains are not suitable for this purpose, for various reasons. A spontaneous mutation in the tyrosinase gene in a C57BL/6J congenic line at the Jackson Laboratory allowed the development of an albino strain that is otherwise identical to C57BL/6J. However, blastocyst yields from this albino strain have been very poor in our hands.  The other albino strain that is commonly used for injection of C57BL/6-derived ES cells is BALB/c, which is notorious for its poor response to superovulation.

Breeding Chimeras

The contribution to the germline by the ES cell lineage in any given chimera is unknown. On average, weaker chimeras are less likely to have ES cell-derived gametes. Nevertheless, a high degree of chimerism is not a guarantee of germline transmission. Therefore, unless the number of chimeras is very large, it is generally in the client’s best interests to breed all of the chimeric males. We suggest producing at least 40 pups from any given chimera before concluding that it will not go germline, and it may be necessary to produce many more than that before the first agouti pup is obtained. One chimera bred by the TMF produced its first ES cell-derived pup after producing 114 other pups !

When deciding which strain of mice to use as mates for your chimeras, consider the strain from which the ES cells were derived, and the genetic background that you eventually want to achieve. Crossing the chimeras with the parental strain of the ES cells allows one to obtain a pure inbred line in one generation. However, 129 lines are often difficult to breed and not all 129 lines are true inbreds. If you want to move your mutation to a background other than the parental strain of the ES cells, it will be necessary to backcross to the desired background for 10 generations to achieve a true congenic.

Summary of our Services and Current Pricing: