Services
Transgenic animals are produced by injecting a DNA construct directly into the pronucleus of a newly fertilized mouse egg. Plasmid or BAC constructs can be used. We routinely inject eggs from FVB/N inbred mice, C57Bl6/JxCBA hybrid mice, and C57Bl6/J inbred mice. Other strains are also available – please inquire. In some eggs, the DNA will integrate into the mouse chromosomal DNA before cell division, and will be present in all cells in the mouse including the germ line. The Transgenic Animal Production service consists of injecting each construct into eggs from ten females (hybrid strains) or fifteen females (inbred strains) per day. We suggest starting with three sessions. Twenty to fifty mice will normally be born from the three days' worth of injected eggs. These animals are screened for the presence of the transgene by a polymerase chain reaction genotyping assay. The number of transgenic animals typically varies from two to eight from three sessions. When the founder animals are at weaning age, the mother will be tested for the presence of specific pathogens including MHV, and transgenic founders are transferred to the investigator when the absence of disease is confirmed. Transgenic founders are thus normally received seven to eight weeks after the original egg injections are performed when the mice are four to five weeks old. Transfer of funds to pay for this service is initiated after positive founders are confirmed. If no transgenic founder animals are produced after the requested number of sessions are injected, the investigator will be consulted before injecting further eggs. For all constructs, the investigator must provide the transgenic construct prepared by a suitable protocol, as well as a set of oligonucleotides primers suitable for PCR genotyping by our Universal Genotyping Assay. Primers for this assay must be 30 nucleotides in length, have 40-60% GC content, have non-homologous 3’ ends, contain no repetitive sequences, and produce an amplimer of 100-400 base pairs in length. We are happy to pick the primers if you provide the transgene sequence, or you can see the Universal Genotyping Assay for more information on primer design. The construct submission request form is found on our request forms page.
Chimeric mice are produced by injecting embryonic stem cells into the cavity of a mouse blastocyst. Chimeric mice resulting from injected blastocysts will be composed of tissue derived from the inner cell mass of the host blastocyst as well as the embryonic stem cells. It is possible for embryonic stem cells to contribute to all cells of the chimeric mouse, including the germ cells. Typically, chimeric mice are used to produce animals derived entirely from embryonic stem cells that have been manipulated in culture to contain a targeted mutation. The chimeric mouse production service consists of injecting embryonic stem cells provided by the investigator or the Siteman Cancer Center ES Cell Core http://escore.im.wustl.edu/ into blastocysts harvested from fifteen females per session. We suggest a minimum of two injection sessions per construct. Twenty to thirty live mice are normally born from this number of injected blastocysts. Normally, the skin color of the mice from which the host blastocysts are derived is different from that of the strain used to produce the embryonic stem cells. Typically two to six mice will have skin and hair with greater than seventy percent ES cell contribution, indicating a good chance for embryonic stem cell contribution to the germline. When the chimeric animals are at weaning age, the mother will be tested for the presence of specific pathogens including MHV, and chimeras are transferred to the investigator when the absence of disease is confirmed. Thus, chimeric animals are typically received seven to eight weeks after the original blastocyst injections are performed when the mice are five to six weeks of age. Transfer of funds to pay for this service is initiated after transfer of the chimeric animals to the investigator. If no chimeras are produced after injection of blastocysts harvested from the requested number of sessions, the investigator will be consulted for potential problems before injecting additional blastocysts. For all constructs, the investigator must provide embryonic stem cells proven to be pathogen free, or we can coordinate directly with the Siteman ES Core to obtain MAP test results. A form to complete before cell submission is found on our request forms page.
The husbandry service includes all aspects of maintaining mice. Breeding, weaning, mouse monitoring, and tail biopsies are performed according to a standard operating procedure that has been used for 15 years with excellent results. All information entered into a commercial computer database. For added protection, specific pathogen screening of MGC colonies is performed twice as often as the standard Division of Comparative Medicine testing for other Washington University colonies. We also offer ancillary services such as PCR genotyping, injections, urine and blood collection, and dissections. Each experimental colony or group is independently tracked, and a weekly report of inventory and procedures is provided. Matings, pregnancies, due dates, births, and a list of adult animals are included in this report. Frequent contact with the investigator is maintained by Email, FAX, phone, and meetings as required. Husbandry charges are calculated as a cost for each time a cage is handled, and is in addition to per-diem cage costs charged by DCM.
We perform genotyping by a Universal Polymerase Chain Reaction Assay, including screens for induced mutations such as transgenes and 'knockouts'. For breeding animals, tissue for genotyping is derived from tail biopsies taken at 5-7 days of age, and assay results are normally obtained by two weeks of age. Tail tissue can be harvested at any age. The price includes up to twenty-five samples tested with our PCR method and primers provided by the investigator. Primers for the Universal Polymerase Chain Reaction Assay must be 30 nucleotides in length, have 40-60% GC content, have non-homologous 3’ ends, contain no repetitive sequences, and produce an amplimer of 100-400 base pairs in length. We are happy to pick the primers if you provide the appropriate sequence, or you can see the Universal Genotyping Assay for more information on primer design.
Targeted mutations in one genetic background (for example, 129SV) frequently need to be transferred to another background more suitable for physiological analysis (for example, C57Bl6/J). This process takes at least ten generations, but strain-specific polymorphic markers can be utilized to speed this process to no more than five generations. Offspring from each round of mating that have the greatest contribution of chromosomes from the desired background are selected for the next mating. This selected breeding process is termed "speed congenics." The core uses single-nucleotide polymorphisms to distinguish between strains, and conversion between most inbred lines can be accommodated. The five generations take approximately 16-18 months to complete moving a mutation from one inbred strain to another. The investigator provides a single female animal and at the end of the conversion will be returned at least one male animal in the recipient background.
Mice that have been exposed to pathogens can be made free of disease through the rederivation service, which is offered in conjunction with the Division of Comparative Medicine. The infected mice are mated, and embryos are surgically removed the next day by Division of Comparative Medicine personnel. These embryos are then surgically implanted into a pseudopregnant recipient in the Mouse Genetics Core, and the pregnant females transferred to the Division of Comparative Medicine quarantine facility. The offspring are tested for pathogens before being released. This procedure is most likely to succeed if several males can be simultaneously mated with wild-type females.
Cryopreservation of mouse embryos is offered to "store" lines of animals not currently needed, to reduce cage costs and cage space, or to provide secure stock in case of contamination. Mice are mated, and embryos surgically removed 2.5 days later and frozen by vitrification. Embryos vitrified and stored under liquid nitrogen can be thawed and develop into live animals at least three years after freezing. The price for this service includes cryopreservation of embryos harvested from 20 donor wild type females and a test thaw and reimplantation. This service requires 5-10 males for mating with females for embryo harvest. We suggest a minimum of 300 embryos be frozen to ensure recovery of the line.
Ovarian transplants are offered to maintain lines with normal ovaries but a phenotype that precludes breeding. The price includes transplant in up to four recipients from one donor female, which must be immunologically compatible with the donor.
Vasectomies are performed on any strain, and males are available for mating ten to twelve days after surgery. This price is per animal.
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