Assistance with transgenic vector design
Distribution of plasmids useful as transgenic vector backbones
We currently have vectors containing combinations of:
- Neo cassettes
- TK cassettes
- DTA cassettes
- pPGKneoF2L2dta- Neo flanked by both Frt and LoxP sites, with Diptheria Toxin A
(Addgene 13445 for map and sequence)
Gene Targeting in ES cells
To make a knockout/knockin mouse with the Mouse ES Cell and Transgenic Facility, MIT investigators provide the targeting construct as well as proof of a viable screening strategy, and the facility will electroporate the DNA into ES cells, perform drug selection, subclone ES cell colonies, and prepare genomic DNA for screening. The ES cell facility then coordinates with the Division of Comparative Medicine for the injection of targeted clones into blastocysts, which occurs in a pathogen-free barrier animal facility. (click here for a flowchart and timeline)
Standard targetings produce only 1%-2% positive clones, so it is necessary to pick approximately 300 colonies to increase the likelihood of obtaining enough correctly targeted cells. Some targetings are directed to loci with high recombination rates (ROSA26 at 50%-75%, for example) and these require fewer colonies for success.
Culture of ES cells and preparation for blastocyst injection
Targeted ES cells or ES cells containing gene traps will be expanded, tested for pathogens, and prepared for injection into appropriate blastocysts.
Preparation of cells for pathogen testing (pathogen tests are required for all imported cell lines before they will be injected). Up to 5 lines can be pooled together for testing.
Injection of DNA, ES cells, or virus into pre-implantation embryos
MIT's Division of Comparative Medicine (DCM) and the KI pooled their efforts, resources, and personnel in the late 1990's to provide a full range of services for the needs of the MIT mouse community.
Requests for ES Cell-based services are processed through iLab.
To Request a Pronucler injection (PNI) of DNA, CRISPR/Cas9 components or virus into fertilized mouse eggs, please click here.
For CRISPR PNI, we have stock of Cas9mRNA from TriLink BioTechnologies available. Please email us if you are intrested in an aliquot.
In addition to the injections services offered through the support of the KI, other services such as cryopreservation of sperm and embryos, IVF, and mouse genotyping are also available. Please see the DCM website for more details, then Login with Touchstone (MIT Certificates Required) to access Transgenics and for the DCM forms needed for these services.
Provide tissue-specific Cre mouse lines
A collection of useful strains of mice are maintained by this Facility and are available to MIT researchers. These strains include Cre mice, FLP mice, Cas9 mice and important wild type strains that are not otherwise available for direct import to the 68N facility. Please see below for detailed fees and ordering instructions. As with all our services, we are not able to distribute the mice outside of MIT.
Provide methods, assistance and training in embryo and mouse analysis
Training options include, but are not limited to:
- Culture of ES cells or iPS cells
- Generation of new ES cell lines
- Isolation and culture of blastocysts
- Generation of MEFs
- Isolation of mouse embryos for histology or other analysis
- Differentiation of ES cells to specific tissue type
- Optimizing breeding conditions for mice
Provide reagents needed for ES cell or other issue culture
- Vials of 3x10`6 irradiated MEFs for use as feeders
- Vial of p1 MEFs
- Vial of germline-proven ES cells
- Media, Serum, and ESGRO for ES cell culture
Other services are also available on a limited basis, please inquire to the ES Core Staff (firstname.lastname@example.org) if you are interested.
Mice available from the KI mouse repository
Updated Jan 2019
All mice listed are housed in the super-clean, barrier facility and can be transferred to any animal facility on the MIT campus. These colonies are maintained at a minimal level to reduce cost and it may take a few weeks to provide more than 4-5 mice of the same gender for a strain. We are happy to build up a colony if you need more, but let us know so that we can plan ahead.
To order mice, please create a request in iLab.
|Mouse Strain||Expression profile||background||Jax strain or Ref.|
|TPA-/-||NeoR, Great for MEFs
|mixed, black coat||Carmeliet et al. 1994|
|CAG-EGFP||bright GFP beginning
in early embryogensis
|Wild-Type Mice Available for Backcrossing|