BREEDING
FOUNDERS FROM MICROINJECTION
1) 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 weeks of age will mate with one male.
2) The GTTF will use wild type littermates generated
from the same microinjection for mating.
3) Since pronuclear microinjection results in random
integrations, the GTTF strongly recommends performing a Southern on
founders to determine copy number and number of integration sites.
4) If multiple integrations sites are found, Southern
analysis should also be performed on the F1 litters to determine the
segregation pattern of the transgene.
5) In order to preserve the transgenic line the GTTF
will retain two Tg/+ males and two Tg/+ females for future breeding.
6) The remaining mice will be available to the investigator
for experimental use or to characterize the line.
EXPANSION OF
TRANSGENIC LINE (Establishing the line)
1) Heterozygous breeding will be set up according to
the investigators' requests and needs. A breeding cage can contain 1
male and 2 female mice. The females will be separated when they are
obviously pregnant.
2) The GTTF will always keep 1 cage of breeding age
transgenic males. All other mice of the same line will be available
for experimental use.
3) 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.
ESTABLISH
HOMOZYGOUS TRANSGENIC MOUSE LINES
1)
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.
2)
Test Homozygousity
a. Possible homozygous mice will mate with wildtype
mice to test for true homozygosity.
b. 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.
c. 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.
d. 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.
3)
Breeding homozygous mice
a. When a pair of mice is identified as homozygous,
the breeding will be set up to generate homozygous mice.
b. The GTTF will keep 1 cage of males and 1 cage of
females. The reserves will be maintained at breeding age.
c. All other mice generated from these crosses will
be available for experimental use.
CHIMERIC
MICE BREEDING
1) A chimeric mouse will mate with one wildtype CD-1
female at 8 weeks of age to test germline transmission.
2) When germline transmission chimeras are identified,
the breeding will be set up according to the researcher's goals:
a. To generate 129Sv/CD-1 knockout mice: A 129Sv chimeric male
will mate with CD-1 female mice.
b. To generate 129Sv inbred knockout mice: A 129Sv chimeric
male will mate with 129Sv females.
c. To generate congenic knockout mice: chimeric male
will mate with inbred strain mice chosen by the investigator.
CD-1 females used in breeding will be provided by the
GTTF. The inbred strain female mice will be purchased at the investigators'
expense.
3) Female chimeras will be mated only when there is
no germline male chimera from the same line or upon request of the investigator.
4) Those chimeras that did not go germ line will be
sacrificed when germline transmission chimeras from the same line have
been obtained or after three white litters.
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