Detailed Project Information

Transgenic Projects

Information and materials you need to provide prior to scheduling microinjections:

• Microinjections are scheduled on a first-come basis and animals are ordered only after DNA has been tested and all information is received
• Investigators must have an NUACUC approved animal protocol describing the use of animals generated by the TTML before microinjections can be scheduled
• Investigators from CMRC must also provide a copy of your current Institutional Biosafety Committee approval letter
• A completed request form including the information listed below is required; submit forms to TTML@northwestern.edu with:
  • Linear maps of transgenic construct
  • Photo of microinjection DNA gel showing a sharp, single transgenic fragment band of the correct size
  • Evidence of a screening assay sensitive enough to detect a single copy transgene
• Purified microinjection quality DNA

Request forms

• Generation of transgenic lines (Word)
• Generation of transgenic embryos: transient transgenesis (Word)
• Generation of BAC transgenic lines (Word)

Microinjection DNA

• For generation of transgenic lines/embryos:
  • 20µg of linear high quality (ultrapure) microinjection DNA (Protocol) diluted in buffer supplied by TTML at a concentration in excess of 40µg/ml
• For generation of BAC transgenic lines
  • High quality BAC DNA (Protocol) analyzed by pulse-field gel electrophoresis for purity and concentration, diluted in buffer supplied by TTML at a concentration of ~50µg/ml

Once this information is received, we will:

1. Assess the quantity and purity of your DNA
2. Schedule and perform microinjections: ~150-200 single-cell embryos will be microinjected initially. This is usually enough to yield 20-25 pups for genotyping or 5 females at the desired day of gestation for transient transgenesis projects
3. Provide tail samples for genotyping: pups from the resulting injections will be weaned at 2 weeks of age and tail samples will be given to you for genotyping (screening for integration)

Expected results

• Transgenesis rates of between 10-25%, or 2-7 founders, are expected for most constructs
• If no founders are identified, a second set of injections will be performed
• Consultation and Southern blot analysis (if PCR is used for initial transgenesis screen) is required, however, prior to a second round of injections

What's expected of you following microinjections

1. Isolation of DNA from tail samples. (Protocol)
2. Genotyping: genotyping results are expected within 2 weeks 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, is requested prior to the transfer of animals.
3. Transfer of animals: once founders have been identified, TTML will initiate the paperwork for the transfer of these mice to your animal room and protocol. Your signature is required before CCM will transfer the mice.
4. Breeding of founders: breeding and subsequent experiments are, of course, your responsibility. Our staff, however, can provide consultation on animal handling, breeding and housing, if necessary.

Time-table

• Received DNA is evaluated, generally takes 1-2 weeks (Assessment of Microinjection DNA)
• Microinjections are scheduled and mice ordered
• Week 1: microinjection and surgical transfer of injected embryos
• Week 2: recipients weighed 10-12 days post-surgery to determine pregnancy
• Week 3: pups born 19 days following surgery
• Week 6: pups weaned, ear tagged and tailed at 3 weeks of age
• Week 6-8: transgenic founders transferred to PI
• Week 9: 6 week old transgenic females founders sexually mature
• Week 11: 8 week old transgenic males founders sexually mature

Project updates-we will notify you regarding:

• DNA assessment
• Scheduled injection date(s)
• Pregnancies and expected due dates
• Number of pups born, date of birth and expected wean date
• Tail sample pick up
• Transfer of pups to your animal room

Costs and Services

• Transgenic and BAC Transgenic Projects
• Transient Transgenesis Projects