Other Services

Design of PCR-based screen strategies: Investigators can have a PCR-based screening strategy for a transgene or ES cell homologous recombination targeting event designed and tested for them.  Ideally, a transgene will have DNA elements arranged in a manner that will allow the design of a PCR method that amplifies a band size that is otherwise not produced using mouse genomic DNA as a PCR template (e.g. the use of a mouse gene promoter in front of a reporter gene).  Investigators would preferably provide a request for service with the DNA sequence of the transgene for PCR primer design (or otherwise database accession numbers and/or gene symbols for transgene DNA elements), a transgene map and a sample of the transgene construct BEFORE submitting a transgenic mouse application form.  ES cell homologous recombination screening strategies will be designed to employ one PCR primer to a targeting construct selection element (e.g. neomycin resistance cassette) and a second primer just outside the shorter arm of homologous DNA so that only homologous recombinant clones will produce a PCR product of the expected size.  This will undoubtedly require ‘long-range’ PCR to span the probably large distance (usually 2-8 kb) between the two PCR primers.  A second PCR screen to likewise confirm homologous recombination at the other end of the targeting event can also be designed and tested for an additional fee.

 

PCR-based screening: Besides designing and testing a PCR-based screening assay for investigators, the Core can also conduct the screen after first isolating DNA from samples, either ES cell clones or mouse tail clippings (see below).  Investigators may need to have screens performed on multiple occasions depending upon the nature of the service they have requested (e.g. transgenic founder mice screening, ES cell clone screening, ES cell homologous recombinant confirmation, and germline transmission confirmation).

 

DNA isolation: regardless of whether or not investigators employ the PCR-based screening service of the Core, investigators can have DNA samples prepared for them from either ES cell clones or mouse tail clippings, charged based on the number of samples to be processed.  The Core can also process plasmid-based targeting vectors prior to ES cell transfection and gel-purify plasmid-based transgenes, free of the plasmid backbone, prior to microinjection, in both cases using MAXIprep-quality plasmid DNA provided by the investigator.

 

Embryo cryopreservation: Freezing mouse embryos is a convenient way to preserve important mouse lines for possible future use or to protect lines against loss due to infection, lab accident, etc.  Embryo freezing could also result in substantial reductions in housing cost and cage use.  The service can be designed to suit the needs of each investigator.  However, a general scheme involves 2-5 breeding pairs of mice, or at least 2 young adult males, to generate enough embryos for each freezing set.  Where hemizygous transgenic males are provided, up to 20 wild type females will be super-ovulated for timed mating with the males.  A minimum of 400 healthy embryos will be frozen per transgenic/knockout line that is hemizygous for the transgene.  In the case where the mouse line is homozygous for the transgene, 250 embryos will suffice.  Several weeks after the completion of the freezing process, a few embryo samples will be removed and processed through thawing and monitoring development to assess their viability and ability to develop to term.  High efficiency of embryo freezing will depend primarily on the reproductive level of the breeding pair since low mating frequency and low percentage of fertilization will lead to a prolonged process.

 

Mouse rederivation:  Our capacity to rederive mouse lines that are known to be carrying infectious agents that are not permitted into our vivarium by Lab Animal Services policy is limited, but we have on occasion worked with investigators and Lab Animal Services to identify mouse housing space that will allow Core personnel to super-ovulate appropriate females for timed matings and collect embryos for transfer to pseudopregnant fosters.  Contact the Core by e-mail to discuss your needs.

 

Long-term liquid nitrogen storage: A number of investigators chose to take their frozen mouse embryos into their own storage, or share them with other laboratories.  Those who chose to leave their frozen embryos in the care of the Core can do so at a cost of $50 per ‘straw’ per mouse line per year, billed and payable each year in early January.

 

Transfer of ES cell clones: As mentioned in the application form for ES cell clone/chimera generation, homologous recombinant clones produced in the Core are held by the Core but investigators can purchase individual clones for further research and distribution to others by signing a standard material transfer agreement and paying a nominal fee.