Phase II: Production of Chimeric Mice

Phase II: Production of Chimeric Mice

In Phase II, potentially targeted clones generated in Phase I are thawed from the 96 well plates, expanded, and frozen for stable long-term storage in LN2. Expanded clones confirmed as correctly targeted by the investigator are scheduled for blastocyst microinjection. Targeted ES clones are microinjected into blastocysts to generate chimeric mice.

Phase II: Expansion and confirmation of targeted clones

• Review of initial screening data with investigator
• Thaw up to 5 potential targeted clones generated in Phase I: expand clones and freeze in LN2 for stable long-term storage
• Cells from each expanded clone will be provided in a single well of a 12-well plate for DNA isolation (Protocol) and confirmation of targeting.
• Review secondary screening data with investigator
• Blastocyst microinjections are scheduled following data review.

Phase II: Blastocyst injections

• 2-3 clones are initially injected into approximately 24 blastocysts per clone.
• Injections generally yield ~5 highly chimeric males.
• If chimerism is low to moderate, another one or two targeted clones are injected.

Phase II timeline:

• Week 1: order mice for blastocyst injections
• Week 2: mice arrive; prep for embryo production
• Week 3: blastocyst injections/transfers
• Week 5: pups born
• Week 8: 3-4 week old chimeras transferred to investigator
• Week 12: begin breeding chimeric males
  • separate/singly-house 7 week old chimeric male for one week
  • introduce 6 week old wild-type females to sexually mature singly-housed males (mating protocol)

Phase II project updates- we will notify you regarding:

• Scheduled blastocyst injections
• Birth of litters and initial impressions
• Percent chimerism of weaned pups and transfer date

Investigator responsibilities for gene targeting projects, phase II:

• Transfer of animals: once pups are weaned, TTML will initiate the paperwork for the transfer of these mice to your animal room and protocol.
• Breeding of founders: breeding and subsequent experiments are, of course, your responsibility. Our staff can provide consultation on animal handling, breeding and housing, if necessary.

Phase II- Frequently Asked Questions

• How are chimeric animals generated?
• Are all ES cell clones capable of generating chimeric mice?
• What is meant by "a highly chimeric mouse?"
• What does the coat color chimerism tell you about the animal? (What is the significance of coat color chimerism?)
• Are all chimeric mice capable of transmitting the mutation though the germline?
• What strain do I breed with my chimeras to generate heterozygous mutants?
• Mating schemes for 129-derived chimeras
• Mating schemes for B6-derived chimeras
• Do I have to continue using Southern Blot analysis to genotype the F1 pups?
• How do I go about mating my chimeric mice?
• Should I breed chimeric females?

Costs and Services

Gene Targeting Main page / Phase I: ES Cell Gene Targeting