TAC logo Biomolecular Research Facilities
University of Missouri-Columbia

Transgenic Animal Core (TAC)

As of March 1, 2005, the TAC has worked with 13 constructs and generated over 100 transgenic pups.
Embryonic Stem Cell Targeting

The Transgenic Animal Core offers:
-- Assistance with designing of targeting construct
-- Electroporation of targeting construct DNA into ES cells and isolation of individual ES clones utilizing appropriate drug selection
-- Expansion of selected ES clones for storage and for screening of correctly targeted clone
-- Expansion and preparation of ES cells for blastocysts injections
-- Blastocyst injections
-- Embryo transfer
-- Health monitoring and weaning of chimeric pups

Investigator has the option to choose full package of TAC services or get help with only specific steps to generate knockout mice.

PLEASE NOTE: If an investigator chooses to use an ES cell line from resources other than the TAC, evidence of infectious disease screening must be provided. Each cell line received into the TAC should be pathogen free. If such testing has not been performed, the investigator is requested to have such testing completed before submission to the TAC. IMPACT IV profile would be sufficient to satisfy this requirement.

Investigator will be required to:
-- Perform cloning procedure to make targeting construct
-- Provide linearized construct DNA for electroporation
-- Establish methods to screen ES cell clones
-- Identify correctly targeted clone by Southern blot and/or RT-PCR
-- Breed of chimeric mouse in order confirm germ line transmission and to establish knockout line

Currently available ES lines:
-- 129 R1
-- C57BL6
-- BALB/c

Please contact the TAC if your project requires a different cell line. The TAC will assist with acquisition of other cell lines as needed. This may result in an additional fee.

Knock-out database
http://research.bmn.com/mkmd
This database provides information on knockouts generated by other investigators.

Use of Gene Trap ES Cell Lines
Before beginning to design targeting construct, the TAC recommends that the PI consider an option of using ES lines from library obtained by gene-trap approach. Thousands of ES clones generated by consortium of research groups (Bay Genomics) are readily available to the scientific research community for a nominal cost. Gene trap approach is based on random integration of reporter vector that contain a splice-acceptor site upstream of the promoterless b-galactosidase gene (lac Z) and neomycin resistance gene. Integration of such vector downstream of functional promoter results in generation of a fusion transcript between endogenous gene and lac Z gene. The expression of fusion protein can be monitored by visualizing of lac Z activity. The insertion also likely inactivates endogenous gene. The use of such "ready to go" ES clones will save 6 to 12 months of work depending on the experience of the PI and his/her team.

However, this approach has some pitfalls, which are important to mention. One problem is that insertional mutagenesis has the potential to undergo splice-around event rendering the integration completely silent. Another problem arises due to the fact that random integration might not disrupt the gene of interest but rather produces a fusion protein. The mutation might not be available on desirable genetic background. BayGenomics used 129/Ola ES line to make their mutant lines. Investigators interested in exploring this option should contact Natalia Karasseva (karassevan@missouri.edu).

Assistance with design of targeting construct
The TAC will be glad to assist with design of constructs for investigators who choose to produce their own. Plasmids with genetic elements for knock-out and knock-in constructs are available from the TAC for research purposes only upon request.

Electroporation of targeting construct DNA into ES cells and isolation of individual ES clones utilizing appropriate drug selection
Standard protocol used by the TAC requires 25-50 micrograms of linearized DNA per one electroporation. Investigor will be asked to provide 100-150 micrograms linearized DNA to perform multiple electroporations. Several rounds of electroporations will be performed to generate approximately 200 ES clones. The purity of DNA is critical for successful electroporation. The DNA should be purified using Qiagen kit #12143 or #12362. Subsequent heat inactivation of restriction enzyme, ethanol precipitation and dissolving DNA in sterile molecular biology grade water (concentration 1 mg/ml) should yield a preparation of sufficient quantity.

Expansion of selected ES clones for storage and for screening of correctly targeted clone
Selected clones will be expanded into 24-well plate format and transferred to investigator for genomic DNA purification and identification of correctly targeted clone by Southern blot and/or RT-PCR. Master plates will be stored at -80C. Since these conditions are not optimal for lon term storage of cells, it is important to complete screening in a timely fashion.

Expansion and preparation of ES cells for blastocysts injections
After clones are identified, 2-3 ES clones will be expanded. At this step additional Southern blot to confirm identified clones is highly recommended. Expanded cells will be prepared for injection into 50 blastocysts.

Embryo transfer, health monitoring and weaning of chimeric pups will be done according to ACUC protocols.
Injected blastocysts will be transferred to appropriately times pseudopregnant recipients. Litters will be monitored and evaluated at weaning for gener and chimerism. Male pups with highest contribution from ES cells (identified by coat color) will be transferred to investigator upon reaching weaning age (3-4 weeks) and after completion of health monitoring. Unfortunately, the Core cannot guaranteee that ES cell clones will produce germline chimeras.

After completion of the project, the investigator will be asked to indicate at publication that this service was done by the MU TAC.

Fees
The production of a knockout mouse requires work over an extended period of time. This work will be billed as vairous phases of the work are completed. The investigator has the option of having the TAC perform all of the ES cell culture work and the blastocyst injection work. Blastocyst injection service is available for investigators who are doing their own ES cell culture work or for individuals who are working with ES cell lines from Gene Trap banks. Interested investigators should contact TAC director Beth Critser (critsere@missouri.edu) to discuss details of knockout fees.