Recombinant DNA Experiments
Because pronuclear injections, CRISPR targeting and ES cell electroporations utilize unique DNA sequences that recombine into the mouse genome, the principal investigator needs to provide the GEMF with an approval number from the Institutional Biosafety Committee (IBC) prior to beginning these experiments.
Prior to the production of any genetically modified mice, and to performing rederivation, archiving or assisted reproduction (IVF), all investigators are required to obtain approval for animal research from MD Anderson Research Compliance by obtaining a MD Anderson Animal Care and Use Form (eACUF). The GEMF cannot initiate any animal experiments without a valid animal use protocol number.
All information related to the experiment will be held fully confidential by the GEMF. No knowledge related to the experiment will be shared unless otherwise approved by the principal investigator.
Submission of a Project Request Form with approved chart string numbers to charge is required prior to the initiation of service by the GEMF. This form will be submitted for billing at the time service is performed, regardless of the final outcome. Please note that most procedures are not guaranteed.
The MD Anderson Genetically Engineered Mouse Facility is supported, in part, by a Cancer Center Support Grant from the National Cancer Institute. Therefore, all publications resulting from the services provided by the GEMF must cite the facility in the acknowledgement section. A copy of all publications resulting from animals created by the GEMF should be submitted to Jan Parker-Thornburg (Department of Genetics, Unit 1010).
Service will be scheduled upon the receipt of all pertinent forms and documentation.
Specific Policies for Experiments
ES cells provided to the facility will be injected after visual inspection of their morphology. Cells that are in poor condition will not be injected, and investigators may be charged an additional fee for blastocysts collected. To avoid unnecessary animal use, we request a week's notice for cancellation of an injection date. For cells produced by the GEMF, we will substitute in an alternate clone for injection if the initial clone fails to give chimeras. For cells produced outside of the GEMF, we will only guarantee the injection of 50 blastocysts.
Gene-Targeting in ES Cells
The investigator is responsible for demonstrating a reliable scheme for identifying targeted ES cell clones by PCR and/or Southern analysis showing detection of the 5' and 3' homology arms by providing results from pilot experiments for this purpose. (Call Jan Parker-Thornburg for further information on certifying PCR analysis.) A convincing strategy for identifying mutant alleles must be demonstrated prior to the initiation of a gene-targeting experiment.
Our facility has produced thousands of founder lines by pronuclear injection. For standard DNA constructs, we will generate three DNA founder animals. While DNA can generally be shown to incorporate into the genome, the gene may or may not exhibit expression. It is not always possible to predict whether the transgene will be expressed and the effects of its expression in the mouse. For this reason, the GEMF cannot guarantee transgene expression. The investigator is responsible for demonstrating a reliable scheme for identifying detection of the transgene at a single copy level by providing results from pilot experiments for this purpose. The GEMF will provide protocols for single-copy detection upon request.
Gene Targeting by Pronuclear Injection Using CRISPR/Cas9, or other Gene Editing Nucleases
The GEMF will inject the components for CRISPR/Cas9 or other gene editing endonucleases by pronuclear injection of mouse embryos to generate gene-targeted animals. We will perform three injection days of supercoiled plasmids for gene knock-in projects, three electroporation days for gene knock-in projects using small (<500 bp) oligonucleotides, and two electroporation days for gene knock-out projects. We require that the components for the procedure are produced by an approved company (contact us for recommendations). We cannot guarantee pups or targeted animals. Please contact Jan Parker-Thornburg prior to initiating a project for details that will help with project success.
While embryo cryopreservation is a valuable tool for conserving mouse resources, certain mouse strains are not amenable to the rigors of the technique. For that reason, it is imperative to know the genetic background of the strain to be cryopreserved. The GEMF will thaw one straw of frozen embryos and culture overnight to the blastocyst stage to insure that embryo freezing is feasible for that particular mouse line.
For sperm cryopreservation, the investigator will be given a schedule for pre-mating the males to encourage new sperm production. Frozen sperm from each male will be tested by IVF for its fertilization capacity. A project is considered complete if the fertilization rate equals or exceeds 20% for each male. A project is considered complete if the fertilization rate equals or exceeds 20% for each male.