Breeding Founders from Microinjection

• Founder mice breeding will start when they reach maturity.
  • A male founder at six to eight weeks of age will mate with two females.
  • A female founder at four to six weeks of age will mate with one male.
• The CMGM will use wild type littermates generated from the same microinjection for mating or mice from vendors if requested.
• Tail snips or ear notches will be given to the investigator for genotyping analysis.
• F1 mice from Indels or Ki/+ founder mice from CRISPR microinjection should be sequenced to confirm gene editing.
• In order to preserve the transgenic line, the CMGM could retain two Tg/+ males and two Tg/+ females for future breeding.
  • The remaining mice will be available to the investigator for experimental use or to characterize the line.

Expansion of Transgenic Line (Establishing the Line)

• Heterozygous breeding will be set up according to the investigators' requests and needs. A breeding cage can contain one male and two female mice. The females will be separated when they are obviously pregnant.
• The CMGM will always keep one cage of breeding age transgenic males. All other mice of the same line will be available for experimental use.
• Upon the request of an investigator, the wildtype mice will be culled if they are not to be used for experimental purposes. These wildtype mice can be used for breeding within the same line. Individual investigators may choose to purchase wildtype mice for breeding at their own cost.

Establishing Homozygous Transgenic Mouse Lines

Breeding to Homozygousity

• Tg/+ X Tg/+ mating will be set up, two pair for each line.
• Litters will be screened for homozygous transgenic mice by the investigator.

Test Homozygousity (If you can't distinguish by pcr)

• Possible homozygous mice will mate with wildtype mice to test for true homozygosity.
• To reduce the cost CD-1 or other wildtype mice can be used for these test matings. Investigators can also order wildtype mice of the same strain at their own expense.
• The pups from these crosses will be sacrificed at weaning and tails snips will be screened. Litters from these crosses can be kept at the request of the investigator.
• A mouse with more than ten offspring that are all Tg/+ will be considered as homozygous. If any of the offspring are +/+ the parent is a heterozygous mouse and will be euthanized as long as the line has sufficient breeders.

Breeding Homozygous Mice

• When a pair of mice is identified as homozygous, the breeding will be set up to generate homozygous mice.
• The CMGM will keep one cage of males and one cage of females. The reserves will be maintained at breeding age.
• All other mice generated from these crosses will be available for experimental use.

Chimeric Mice Breeding

• A chimeric mouse will mate with one wildtype CD-1 female at six weeks of age to test germline transmission.
• When germline transmission chimeras are identified, the breeding will be set up according to the researcher's goals:
  • To generate 129Sv/B6/CD-1 mice: A 129Sv/B6 chimeric male will mate with CD-1 female mice.
  • To generate 129Sv/B6 floxed mice: A 129Sv/B6 chimeric male will mate with B6 or 129 FLP females.
  • To generate 129Sv/B6 knockout mice: A 129Sv/B6 chimeric male will mate with B6 Hprtcre females.
  • To generate congenic knockout mice: chimeric male will mate with inbred strain mice chosen by the investigator.
  • CD-1 females and either FLP or Hprtcre females (investigtor's choice) used in breeding will be provided by the CMGM. Additional females are purchased at the investigator's expense.
• Female chimeras will be mated only when there is no germline male chimera from the same line or upon request of the investigator.
• Those chimeras that did not go germline will be sacrificed when germline transmission chimeras from the same line have been obtained or after two white litters.