Initiate a Transgenic Project

You will need the following to initiate a project:

• An approved animal protocol, describing the use of transgenic mice generated by the Transgenic and Targeted Mutagenesis Laboratory (TTML)
• A transgenic construct containing all the regulatory elements necessary for gene expression
• Evidence of a genotyping assay sensitive enough to detect a single copy transgene
• Choose a mouse strain for the microinjections
• A completed Transgenic Project request form for each submitted transgenic construct
• A service 'order' request through NUCore for Generation of Transgenic Mice
• Restricted DNA (plasmid-based transgenes) or injection quality BAC DNA

Request Forms

• Generation of transgenic lines
• Generation of transgenic embryos: transient transgenesis
• Generation of BAC transgenic lines

Microinjection DNA

For plasmid-based transgenes, provide restricted DNA for final purification:

• Perform a restriction digest on your transgenic construct to generate ≥50µg of the transgenic fragment
• Run 100ng DNA on a gel to insure the digestion is complete and the bands the expected size
• Inactivate the enzyme when digest is complete
• Take a picture of the gel with the DNA ladder and transgenic band clearly labeled, and band sizes indicated
• Bring the picture and the rest of the digest (in ~100-150µl volume) to the Transgenic Lab (Lurie B217)
• Once the transgenic band is purified, a sample will be provided so that you can:
  • Prepare copy standards to demonstrate the sensitivity of your genotyping PCR
  • Verify that the correct band was isolated

For generation of BAC transgenic lines:

• Provide high-quality BAC DNA (see protocol) diluted in buffer supplied by TTML at a concentration of ~50µg/ml
• We will analyze the quality of your DNA by pulse-field gel electrophoresis and test for toxicity

What's expected of you following microinjections:

• Isolation of DNA from provided tail samples and genotyping (see protocol)
• Genotyping results are expected within one week of receiving the tail samples
• Written confirmation of genotyping results, in the form of an email communication, a photocopy of the Southern blot autorad, or a picture of the PCR gel
• Once founders have been identified, TTML will initiate the transfer of these mice to your animal room and protocol: your online approval is required before CCM will transfer the mice
• Breeding and subsequent experiments are, of course, your responsibility
• Our staff, however, can provide consultation on animal handling, breeding and housing, if necessary