IDDRC Mouse Gene Manipulation Facility Details
1. To obtain instruction from Core personnel or access to Core services, please contact the Manager or the Core Director by phone, email, or in person. Investigators are asked to provide a 200-word description of the project. You will then be contacted by the Core Manager to discuss the most effective ways in which the Core facility can be used to accomplish the research goals. IDDRC investigator use of Core facilities is determined on a first‑come, first‑serve basis. When the schedule permits, the Core provides service to non-IDDRC investigators. The Core always gives scheduling priority to IDDRC users over non-IDDRC users.
2. The generation of genetically altered mice using microinjection technology can be compromised by a variety of factors. These factors include but are not limited to the quality and/or nature of the injected material, individual variation between animals, the conditions of animal housing, and animal pathogens. The IDDRC Mouse Gene Manipulation facility will make the best effort to generate genetically altered animals with the material that investigators supply, but the core cannot guarantee that desired animals will be obtained for the reasons stated above.
3. Proper genotyping assays for all projects are essential to successful projects. Do not underestimate either the importance of these assays or the time it may take to establish working assays before submitting material to the Core. Please see the “GENOTYPING REQUIREMENTS FOR GENE TARGETING AND TRANSGENIC EXPERIMENTS ” section below to review what is required.
4. The core will attempt to inject investigators’ materials in a timely matter, but unforeseen events may require rescheduling of injection dates.
5. The animals used by the IDDRC Mouse Gene Manipulation facility are housed in an isolated room within the Children’s Hospital animal facility (ARCH). While we continually monitor our animal population for mouse pathogens and are currently pathogen-free with the exception of Helicobacter, occasional mouse pathogens are detected within ARCH. The IDDRC facility therefore cannot guarantee pathogen-free animals.
6. Animal housing and any pathogen screening required by the animals that result from injections done on the behalf of investigators will be charged to the investigators. Some housing may be done at Charles River Laboratories.
7. The injection prices are subject to change. At the time of a price change scheduled injections will be conducted at the old rate, but yet unscheduled injections will be charged at the new rate.
8. An injection can be cancelled up to 18 days before a scheduled injection date with no charge. Cancellations fewer than 18 days before an injection date may incur a $250 animal fee.
9. The procedures required for making genetically altered mice are approved in the ARCH animal protocol; 11-04-1939R and the Harvard Committee on Microbiological Safety (COMS) license 01-105. However, the individual lines of mice generated by the IDDRC facility must be registered by the investigator with the Harvard COMS and approved in an animal protocol reviewed by the investigator’s institution’s animal care and use committee (IACUC).
10. Please cite the IDDRC grant # 1U54HD090255 in all papers that report results of experiments on animals and cells generated by the Mouse Gene Manipulation Facility. In addition, please notify the IDDRC Mouse Gene Manipulation facility of such papers prior to publication and give a copy of the final paper to the facility.
11. Please notify the IDDRC facility of the fates of animals and the results of breedings of animals generated by the IDDRC Mouse Gene Manipulation facility.
12. When requested, please submit summaries of experiments performed with the animals produced by the IDDRC facility for the non-competitive and competitive renewals of the IDDRC grant.
GENOTYPING REQUIREMENTS FOR GENE TARGETING AND TRANSGENIC EXPERIMENTS
Successful gene targeting and transgenic procedures are absolutely dependent on perfect genotyping assays worked out before procedures are begun. It takes as much or more effort to get the genotyping assays actively working as it does to make targeting vectors and/or transgenic constructs. Please read the following discussion carefully and then make an appointment with the Core manager to discuss your genotyping strategies.
I. For Gene Targeting
The goal of genotyping is to identify which clones are homologous recombinants that result from the replacement of the endogenous locus with the sequences in the targeting vector. To determine that a clone contains the desired recombination usually requires a set of genotyping assays that rule out other possible molecular recombinations. It is imperative that Southern and/or PCR assay assess proper recombination of both the 5′ and 3′ arms of the targeting vector. This absolutely requires Southern probes or for PCR at least one of the primers in each reaction) that are homologous to DNA sequences not contained in the homologous arms of your targeting vector. These “external” (as opposed to “internal” or within the targeting vector) probes/primers are absolutely necessary for demonstrating unique homologous recombination into the proper locus. Assays that demonstrate homologous recombination of both the 5′ and 3′ arms of the targeting vector usually require sets of probes/primers external to both the 5′ and 3′ ends, but on rare occasions can be done with one probe if the detected band includes the entire targeting vector. Having assays for both the 5′ and 3′ arms is the only way to rule out an insertion/duplication event.
For targeting vectors that contain mutations at a distance from the positive selectable marker (“remote” mutations), a definitive assay to demonstrate homologous recombination including the remote mutation to rule a random integration of the mutation is required. You cannot depend on the remote mutation “tagging” along with the drug selection event.
(For more on genotyping, please see “more on genomic rearrangements in targeting arrangements at the end of this section.)
Before we embark on a project, we require that people demonstrate on DNA from wild type ES cells (we can supply this) that all the planned genotyping assays in fact work. You will need to show us that:
1. Restriction enzymes digest wild type ES cell DNA.
2. Southern probes give a single wild type band on digested ES cell DNA.
3. PCR reactions work at the single copy level in the presence of complex genomic DNA. The positive control plasmid DNA must be titrated down to the number of molecules that are equivalent to the number of molecules of a single copy gene in the amount of genomic DNA used in the assay. These reactions usually involve amplifying several kilobases of DNA. We have seen these work for greater than 5 Kb. Contact the Core manager for information about working out conditions. (You may need to make positive control plasmids for testing PCR reactions – CAUTION – make the control amplified band a detectably different size than your real recombinant band – because when you screen your ES clones, you don’t want to be uncertain about whether you are looking at a real recombinant or contamination with your control plasmid. An alternative approach to making a positive control plasmid involves designing primers that span the neomycin gene- i.e. one external primer and one internal primer in the opposite homologous arm sequence. Demonstration that these primers work on wild type DNA does not guarantee that they will work on the longer homologous recombinant DNA, however). Please note that lox sites will not serve as amplification primer sequences (too AT rich) so it is usually imperative that a novel restriction enzyme site is engineered at a remote lox site or any other remote mutation for that matter.
Beware of repetitive DNA in your targeting arms and probes.
II. For Transgenic Experiments
Southern and/or PCR assays to assess integration of the injected transgenic construct into genomic DNA.
Before we embark on a project, we require that people demonstrate on wild type tail DNA spiked with positive control template that at least one planned genotyping assay works. This can be either a Southern or more likely a PCR assay. For both, the positive control plasmid DNA must be titrated down to the number of molecules that are equivalent to the number of molecules of a single copy gene in the amount of genomic DNA used in the assay.
You will need controls to show that:
1. Any planned restriction enzymes digest wild type tail DNA. (Note many enzymes do not cut sites that have a CG in the sequence due to CpG methylation.)
2. Southern probes give a single wild type band on digested tail DNA spiked with positive control template.
For PCR assays:
You will need controls to show that:
PCR reactions work at the single copy level in the presence of complex genomic DNA. CAUTION – it is often advisable to make a positive control template that will give an amplified band that is a detectably different size than your real integrated band – because when you screen your pup tail DNA, you don’t want to be uncertain about whether you are looking at a real integrant or contamination with your control plasmid. Please note that lox sites will not serve as amplification primer sequences (too AT rich).
MORE ON GENOMIC REARRANGEMENTS IN TARGETING EXPERIMENTS
The following discussion focuses on targeting experiments that include “remote” mutations as defined below, but is relevant to all targeting experiments. “Remote” mutations are desired targeted mutations that are in targeting vectors in addition to the neomycin (or other positive drug) selection cassette and that have genomic sequences between the positive drug selection gene and the mutation.
There are 5 possible genomic integration events that can occur upon transfection/selection and that we will concern ourselves with.
You want only the one (category #3 below/and sometimes #5 for controls) and must design sufficient genotyping assays to determine exactly the cells in which the exact correct/desired event has occurred.
- Random integration anywhere in the genome/not at your desired locus – doesn’t want these and are usually easy to rule out by even just one genotyping assay.
- Insertion of your targeting allele into the desired locus resulting in an imperfect duplication. You get one copy of the wildtype with an adjacent copy of your mutant allele. This can happen on either side of the wildtype locus (5′ or 3′) – don’t want these, and you can be easily deceived by these if you have incomplete genotyping info.
- Correct homologous recombination of your targeting allele resulting in a replacement of the wildtype locus with your targeting allele including your “remote” mutation – these are the ones you want, but they can easily be confused with category 4 (see below).
- Correct homologous recombination of your targeting allele resulting in a replacement of the wildtype locus with your targeting allele NOT including your “remote” mutation but also WITH A RANDOM INTEGRATION somewhere else in the genome of at least the part of your targeting vector with your remote mutation – these clones are probably rare, but you really don’t want the uncertainty that what you think is correct is really one of these. You can easily be deceived into thinking these are category 3 (see above) if you have incomplete and/or improper genotyping assay.
- Same as 4 but with no additional random integration event. These are good wildtype control lines in that they will have the neo gene but not your “remote” mutation. These can be confused with category 3 clones if you do no genotyping for the remote mutation.
When all these events are individually screened by a collection of complimentary genotyping assays then you can be truly sure of your positives.