Services

NIBS Transgenic Research Center Services:

• Pronuclear Microinjection
• Gene targeting
• Blastocyst Microinjection
• ICSI & Sperm Freeze
• Pathogen-free Rederivation
• Consultation Services

Pronuclear Microinjection Services

The Transgenic Research Center (TRC) in Room 412 will inject the DNA into fertilized mouse eggs and transfer them into recipients. The Center will care for the pregnant females and their litters until three weeks after the mice are born. At this time the animals are transferred to the investigator.

Investigators are required to:

1.  Generate a DNA construct and completes the Transgenic Services Request Form prior to contacting the Center.

The following conditions must be met by the investigator prior to the submission of plasmid DNA:

a) A map of the plasmid, delineating the insert and relevant restriction sites.

b) Properly prepared DNA construct following the guidelines from the TRC.

        c)  Evidence of a screening strategy (Southern blot, dot blot, PCR)

d) A photograph of a gel that demonstrates the quality of the transgene DNA fragment and clean separation from vector DNA must be provided.

2. Contact the TRC staff (ext. 8589) to reserve a date for microinjection.

3. Genotype potential founder transgenic mice and establish the line of mice from each founder. The TRC strongly recommends screening potential founders by Southern Blot  and developing a strategy prior to project initiation.

Gene Targeting Services

The investigator’s targeting construct will be electroporated into ES cells. following conditions must be met by the investigwill be frozen and the investigator will be provided with two 96-well replica plates of each stock plate for DNA isolation.

Investigator are required to:

1. Generate the targeting vector and complete the Gene Targeting Services Request Form prior to contacting the TRC.

The following conditions must be met by the investigator for the submission of plasmid DNA:

         a) Provide a detailed map of the targeting vector.

         b) Supply 100μg of linearized DNA at a concentration of 1μg/μl in sterile, DNAse free double distilled water.

         c) Gel showing complete digestion of linearized targeting vector. 

        d) Provide a screening strategy to that distinguishes homologous recombinants from random integrations or the wild type gene.

2. Contact the TRC staff (ext. 8589) to reserve a date to perform the gene targeting service. 

3. Screen the ES cell clones for recombination by Southern Blotting.

Blastocyst Microinjection Services

The TRC personal will inject and transfer blastocysts to pseudopregnant females. The Center will care for the pregnant females and litters for three weeks after birth.

Prior to injection of ES cells into blastocysts, investigators are required to:

1. Complete the Generation of Chimeric Mice Request Form.

2. Provide a Southern Blot that demonstrates homologous recombination in ES cells.

3. Call the TRC staff (ext. 8589) to prepare ES cells for blastocyst injection. 

Intracytoplamic Sperm Injection (ICSI) and Sperm Freeze

ICSI can be used to rapidly expand mouse lines in strains that may be poor breeders or when few breeders are available. Meanwhile, we will provide the services to cryopreserve sperms from two healthy males of the line you requested, store the sperms in our liquid nitrogen freezer, and give you equal vials of sperms to keep in your own location. When you need to use the lines again, we will perform intracytoplasmic sperm injection and generate animals for you.

PIs request to complete the ICSI Request Form, Sperm Freeze Form and provide two healthy males.

Pathogen-free Rederivation

The TRC will rederive lines of mice that have been become contaminated or have been acquired from conventional colonies at other institutions. Prior to mouse rederivation, investigators are required to submit the Rederivation Request Form for services and contacts the Center (ext. 8589) to schedule the rederivation.

Consultation Services

The TRC provides consultation services for the design of transgenes and knockouts, as well as genotypic and phenotypic analyses.  Appointments with TRC personal should be requested via E-mail or by calling ext. 8585.