Services

Assistance with transgenic vector design

Aurora Burds Connor (escore@mit.edu) is happy to help with individual questions for your construct.

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 Preclinical Modeling Facility, MIT investigators provide the targeting construct as well as proof of a viable screening strategy. 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 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 PCM Core Staff (escore@mit.edu) if you are interested.

Mice available from the KI mouse repository

Updated August 2022

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.
       
Wild-Type Mice Available for Backcrossing
Jaxmice C57BL/6j     000664