ES Cell and Transgenics Facility

Overview of Services

 

This shared resource of the Koch Institute provides fee-for-service support to all MIT investigators who utilize novel mouse models to study human diseases such as cancer. Access is available to all members of the MIT community, to the extent permitted by available capacity. Priority access is given to KI members, NCI-funded research projects and other contributing user groups in recognition of funding support.

 

Facility Leadership

 

Technical Director

Aurora Burds Connor, Ph.D.

Ph: 617-452-3658


Project Coordinator

Noranne Enzer

Ph: 617-324-3911


 

Facility emailescore@mit.edu

 

Facility FAX: 617-324-3912


 

Location and hours of operation

Hours Location

Monday - Friday

8:00AM - 4:00PM

Excluding Holidays    

MIT Bldg 76 Rm 195

Kendall Square,

500 Main Street

Cambridge, MA

(See Map below for directions)

Links and Resources

 

  1. Core Facility Webpage: http://ki.mit.edu/sbc/escell
  2. Swanson Biotechnology Center

Services

Name Description Price
    *
1. ES Targeting for New User 

Select this option if you have never generated a successful gene targeting construct before.  You will schedule an initial project consultation with Aurora Burds Connor  and Noranne Enzer through iLab.


Please read the  "Beginner's Guide to Gene Targeting,"  written by Aurora under the Methods section BEFORE your consultation.


Please bring the following to your consultiation:

- gene/locus name and structure

- any relevant publications


 
*
1a. Gene Targeting in pure 129 or 129/B6 hybrid ES cells, 288 clones  

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.

 

Your project will begin with a consultaion.  If you are a new user, and have never done a targeting project with the facility before, please DO NOT select this service - please select a Free Consultation (above) which should occur before you begin building the construct so that the targeting can be scheduled most efficiently.

 

 Please read the "Beginner's Guide to Gene Targeting," written by Aurora Burds Connor under the Methods section BEFORE your consultation.

 The facility will:

  1. Electroporate the DNA into ES cells
  2. Perform drug selection
  3. Subclone ES cell colonies
  4. Prepare genomic DNA for screening.

When correctly targeted clones are identified, the ES cell facility then coordinates with the Division of Comparative Medicine for the injection of clones into blastocysts, which occurs in a pathogen-free barrier animal facility. 


The charge listed includes gene targeting, picking 288 clones, stocking 5 independent clones and preparing 3 clones for injection into blastocysts to make chimeric mice.  The cost may change if fewer clones are required or if more injections are needed. 

 
*
1b. Gene Targeting in pure 129 or F1 129/B6 ES cells - 96 or fewer colonies 

This option should only be selected for loci known to have a very high rate of recombination (ROSA26, Cre or FLP of a clone, or targeting with the Col1a-FLP system - for example)

 

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.

 

Your project will begin with a consultaion.  If you are a new user, and have never done a targeting project with the facility before, please DO NOT select this service - please select a Free Consultation (above) which should occur before you begin building the construct so that the targeting can be scheduled most efficiently.

 

 The facility will:

  1. Electroporate the DNA into ES cells
  2. Perform drug selection, if needed
  3. Subclone ES cell colonies
  4. Prepare genomic DNA for screening.

When correctly targeted clones are identified, the ES cell facility then coordinates with the Division of Comparative Medicine for the injection of clones into blastocysts, which occurs in a pathogen-free barrier animal facility. 

 

The charge listed includes gene targeting, picking a small number of clones, stocking 5 independent clones and preparing 3 clones for injection into blastocysts to make chimeric mice.  The cost may change if fewer clones are required or if more injections are needed.

 
*
2. Gene Targeting in pure C57BL/6 ES cells 

Due to the genetic instability and poor performance of pure C57BL/6 ES cells, we do not recommend targeting them.

 
*
2. Special Project Request 

Please request this service to set up an initial consultation with the ES Cell & Transgenic Core Facility.

Some examples of special projects include generation of NEW ES cell lines from your mice, viral infection of ES cells to introduce a fluorescent marker, generation of chimeras from multiple cell lines.

Inital consultations are Free of charge.

 

 
*
3. 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)
 
*
3. Expand and Stock an ES Cell Line or Clone 

Cells are expanded optimally and 3-5 stock vials are frozen.

DNA will be prepared from a portion of cells for confirmation by the investigator.  Alternately, growing cells can be given to you for a western, nothern or other analysis.

 
*
4. Prepare cells for pathogen testing 

Mouse ES cells arriving from ANY outside source must be tested for pathogens at RADIL, through the DCM Pathology Lab.

The ES cell facility staff will expand the line, make frozen stock, and grow cells without antibiotics for two weeks prior to submitting the cells to DCM for pathogen testing. 

Up to 5 lines can be pooled together for testing to reduce cost of the pathogen test ($470), though individual charges will be incurred for the preparation of each line.

All new mouse lines must be added to your CAC protocol before ES cells can be injected to generate mice, though the cell culture and pathogen testing may occur while you await CAC approval.

 

 
*
5. Prepare ES clone for injection to generate chimeric mice 

The ES Cell Facility will coordinate with DCM to schedule the injection of your ES Cell clone into mouse blastocysts to generate chimeras.

One ES cell clone will be cultured and prepared for each request.  Only one clone is injected each day.

The fee for ES cell injection, care of mice and pathogen tests on mice is charged by DCM.  Chimeras arrive in the investigator's mouse room after pathogen reports arrive, at roughly 6-8 weeks of age.

 
*
Establish New ES Lines from mouse colony 

Timed pregnancies in your colony will allow for harvesting of pre-implatiation embryos, which develop in vitro and are coaxed into becoming ES cell lines.  

New ES lines require karyotyping before they are used for experimentation.  It is not unusual for total costs of this type of project to reach $3,000+.

 
*
Provide assistance and training in embryo and mouse analysis 

Training and Troubleshooting/Advice are provided happily and charged by the hour (price listed is per hour).  Extra charges will be added if media/supplies are needed as part of the training.


Training options include, but are not limited to:

  • Generation of MEFs
  • Isolation of mouse embryos for histology or other analysis
  • Isolation and culture of blastocysts
  • Generation of new ES cell lines
  • Culture of ES cells
  • 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
 
*
Request Cells 

We can provide:

  • Germline-proven ES cells
  • MEFs at p1 for expansion
  • Irradiated MEFs to use as feeders
 
*
Request Repository Mice 

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, and important wild type strains.

We are unable to provide any of our services, including mouse distribution, to non-MIT investigators.

 
*
Special Project 

This project will be designed by the Facility after we consult with you. Please choose a free consultation in the section above, rather than this item.

 
*
129S4/Jae Male    *
Assisted Work    *
C57BL6j Female    *
C57BL6j Male    *
CMV-Cre B6 Female    *
CMV-Cre B6 Male    *
Culture of ES cells and preparation for blastocyst injection    *
Expansion and Stocking an ES Line or Clone 

Cells are expanded optimally and 3-5 stock vials are frozen.

DNA will be prepared from a portion of cells for confirmation by the investigator.  Alternately, growing cells can be given to you for a western, nothern or other analysis.

 
*
FLP B6 Female    *
FLP B6 Male    *
Pathogen Tests 

ES cells imported from outside MIT will be expanded, tested for pathogens (DCM charge of $450, not included)

 
*
Prep & Ship 1-2 Mouse lines for Karyotyping    *
Prep & Ship Additional lines for Karyotyping (in same shipment)    *
Small Targeting, Picking up to 96 colonies    *
Standard Targeting, Picking up to 288 colonies    *
TPA-/- Female    *
TPA-/- Male    *
Training    *
Vial of p1 MEFs    *
Vials of 3x10'6 irradiated MEFs for use as feeders    *