Services

Production of Transgenic Mice

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.


Production of TALENs & CRISPR Mice

Like traditional gene targeting in embryonic stem (ES) cells, the newest technology for inducing double-strand breaks (DSBs) is being utilized to make sophisticated molecular modifications to endogenous genes. By direct injection of DNA or mRNA encoding site-specific nucleases into the one-cell-stage embryo, we can generate DSBs in DNA at specified sequences, leading to targeted mutations. Co-injections of a single-stranded or double-stranded DNA fragment containing homology to the sequences flanking the DSB can produce mutant alleles with precise point mutations or DNA insertions. The production of genetically modified mice has been greatly accelerated by these novel techniques, which also significantly reduce the cost to the investigator. We inject eggs from C57Bl6/JxCBA hybrid mice for this service. Other strains are also available – please inquire. The DSB Animal Production service consists of injecting each construct into embryos from fifteen females per day. We suggest starting with three sessions. Fifteen to twenty-five mice will normally be born from the three days' worth of injected eggs. These animals are screened for the presence of the mutation by a polymerase chain reaction genotyping assay. The number of mutant animals typically varies from one to five 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 founders are transferred to the investigator when the absence of disease is confirmed. 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 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. If you wish to have us screen for founders you will also have to provide 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.


Production of Chimeric Mice

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.


Animal Husbandry

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.


PCR Genotyping

We perform genotyping by a Universal Polymerase Chain Reaction Assay, including screens for induced mutations such as transgenes and 'knockouts' for colonies using our Husbandry Service. 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 one reaction with up to 3 primer pairs (if multiplexing is optimized) using 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.

We require that samples be submitted before noon on Mondays for processing that week. Results will be sent to you by Friday afternoon. Please fill out the genotyping request form and send via email to the PCR lab at: mgcpcr@kids.wustl.edu Please include a hard copy of the form with your tissue samples. Tissue should be submitted in 0.2uL PCR strip tubes. We use VWR tubes, cat. #53509-306. For each litter, please clearly label the first tube with a unique identifier, such as AA, which denotes the litter ID. The rest of the tubes should be labeled with the sample numbers (2-6 etc) on the side of the tube, not the cap, with permanent marker. Samples should be1-3mm in size. Please be sure that you push the tissue sample to the bottom of each tube for the digestion process. Place your strip tubes in an empty pipet box so that the samples remain upright and tails are not dislodged to the side or top of the tube.


Speed Congenics

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.


Rederivation by IVF

Animals that have known health risks must be kept in isolation and quarantine after importation. To reduce the time and increase the safety of the importation, we offer In Vitro Fertilization of imported sperm to generate pathogen free animals that are age-matched. The service requires imported sperm by obtained by the investigator and an IVF request submitted. Typical wait times for IVF recoveries are 3-6 weeks.


Cryopreservation

Cryopreservation of mouse sperm 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. Two male mice are used for donors, sperm is isolated and mixed and then stored in cryo straws under LN2. One thaw and IVF recovery is included in the price. Sperm cryo provides a less expensive option and produces many more chances at recovery along with the ability to obtain many age matched offspring from recovery. Most strains freeze well, inquire for some specific inbred strains. Storage is available at a low annual fee in two separate sites for security.


Ovarian Transplants

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

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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