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UCI > Research > TMF > ES Cell Injection Services ES Cell Injection Services
Introduction:
Similar to DNA microinjection to produce transgenic mice, ES cell injection involves the superovulation and mating of blastocyst donors to obtain large numbers of embryos, and injected embryos are implanted into pseudopregnant foster mothers. Embryos can be transferred to either the uterine horns or the oviducts of foster mothers. ES cells to be injected can be supplied by the client or grown in our facility. Usually, cells are expanded from frozen stock a few days prior to injection, but freshly harvested cells can be delivered to our facility the morning of injection. In the latter case, the client must provide evidence that the cells are free of mouse pathogens. Frozen cells from most outside sources will be sent out by us for pathogen testing, including mycoplasma, at additional expense to the client. When cells are supplied by the client on the day of injection, we would like to receive the equivalent of a reasonably confluent 35 mm plate. If grown on feeder cells, the ES cells must be pre-plated on a gelatinized plate for about an hour (under standard ES cell culture conditions) to allow selective attachment of the feeder cells. The supernatant from the pre-plate should then be centrifuged and the cell pellet, enriched for ES cells, should be resuspended in about 0.3 ml of ES cell media. This suspension should be delivered on ice to our facility. We charge a separate fee when we expand frozen cells for injection. The fee depends on the number of clones being expanded and whether they are grown on feeder cells or not. | back to top |
Ordering:To order this service, go to our on-line order form. After filling out the on-line Service Request Form, print a hard copy, have it signed by the Principle Investigator, and mail or fax it to Tom Fielder. In addition to the Service Request Form, you must submit the following:
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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. | back to top |
Performance Guarantees:We offer two levels of service for ES cell injection. The Basic service guarantees only that we will inject at least 50 blastocysts successfully (i.e., at least 50 blastocysts will survive the injection process and be transferred to pseudopregnant foster mothers). It does not guarantee that we will produce any pups from those blastocysts. The Basic service is usually completed in one day of injection. The Premium service guarantees that we will produce at least 3 chimeras or 50 total pups, whichever comes first. For this service, we schedule 2 injection days to begin with and then wait to see how many pups and chimeras are produced before scheduling another day. | back to top |
Service Description:The Basic and Premium services include
If included on the order, we will also thaw and expand ES cells, freeze back multiple vials, and pre-plate the cells the morning of injection to selectively reduce the number of feeder cells. ES cells from outside sources may need to be tested for mycoplasma and mouse pathogens at additional cost to the client. | back to top |
Identification of Chimeras:Chimeras are usually identified on the basis of coat color (but see below for exceptions). Most 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 are males. Strong contributions by ES cells to the germline can cause a female blastocyst to develop into a chimera that appears to be male. For the purpose of our performance guarantee, chimeras consist of any mice with visible agouti fur, regardless of sex. An exception to the above is when an ES cell line derived from C57BL/6 mice is used. One such line, HGTC8, derived at NIH, is available for gene targeting in our facility. 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 must be genotyped by the client 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 | back to top |
Breeding Chimeras:The contribution to the germline by the ES cell lineage in any given chimera is unknown. On average, weaker chimeras (those with less agouti fur) 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 20 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. 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. | back to top |
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University of California, Irvine
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Privacy & Legal Notice |
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Gene targeting is carried out in mouse embryonic stem (ES) cells in vitro. Successfully targeted cells are then injected into mouse blastocysts to make chimeric mice, whose tissues consist of cells derived from the blastocyst and cells derived from the ES cells. 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. (See below for more information about breeding chimeras.)