基因敲除模式小鼠的饲养与交配

Homozygous
25%
-/-
Heterozygous
50%
+/-
Wildtype
25%
+/+
Simple Breeding Schemes
+/-/-
Heterozygous
Homozygous
Heterozygous
50%
+/-
-/Homozygous
50%
Backcrossing breeding
3’loxP-F/3’loxP-R
Cre-deleter
Δ/+, Cre/+
+/+ ,Cre/+
Cre/Cre or Cre/+
+
+
+
-
+
+
Controls: +/+, Cre/+
Conditional convert to Germline Knockout
Δ/+ Cre/+ Δ/+ Cre/+
WT-F WT-R
E1
E2
E3
E4
Targeted allele(fl-neo)
E1 E2
Frt
loxP-F
LoxP
loxP-R
Frt LoxP
E3
E4
Flox allele(fl)
E1 E2
Mate with Flp-deleter mouse
Frt-F Frt-R
E3
E4
Deleted allele(Δ)
loxP-F loxP-R
Deleted allele(Δ)
3’loxP-F 3’loxP-R
Mate with Cre-deleter mouse
E3 E4
E1
Conditional convert to Germline Knockout
Genotyping strategy
Genotype
WT-F/R
WT-F/R
WT-F/Neo-R
-
+＋/－来自＋ /＋++
+
-
Controls: Wildtype or Heterozygous
Complex Breeding Schemes
Conditional knockout mice
General breeding scheme
Wildtype allele(WT)
Cryopreservation options
Reduce cost
2.progeny testing Controls: wildtype littermates
Complex Breeding Schemes
Germline Knockout mice
Germline Knockout mice
Genotyping strategy
Genotype － /－
Keep the age range of your breeders between two
and eight months old
Retain two generations of a strain, and do not eliminate one until the next one is producing Monitor breeding performance closely Consider cryopreserving a strain
Replace breeders from a trusted vendor each 5 to10 generations Establish a genetic quality control plan Genome scans- SNPs, MITs, and biochemical markers, remove spontaneous mutants Cryopreserve unique strains
Tg/+ Heterozygous
50%
+/+ Wildtype
Controls: wildtype littermates
50%
Breeding Schemes for transgenic mice
Genotyping strategy
1.QPCR Tg-F Tg-R ---Internal Positive Control
Δ/Δ
Δ/+
+/+
Genotyping strategy
Genotype
Δ/Δ Δ/+ +/+
WT-F/R
+ +
3’loxP-F/3’loxP-R
+ + -
Cre-deleter
-
Controls: Δ / +; + / +
Long-Term Colony Maintenance
Long-Term Colony Maintenance
N1
N# = backcross generation number F# = brother X sister generation number
Complex Breeding Schemes
Transgenic mice
Transgenic mice
• Founder mice:
transgenic mice that develop from the injected eggs
Breeding Performance Factors
Hybrid vigor---more, larger, and healthier litters than inbred strains. Strain-specific behaviors---Aggressive behaviors or poor mothers Mutations and transgene effects---Embryonic lethal, or infertility or reduced fertility
fl/fl, Cre/+ fl/+, Cre/+ +/+, Cre/+ fl/+, +/+ fl/fl, +/+
Genotyping strategy
Genotype WT-F/R loxP-F/loxP-R Cre
fl/fl, Cre/+
fl/+, Cre/+ +/+, Cre/+ fl/+, +/+ fl/fl, +/+
+ + + -
+
+ + +
+
+ + -
Controls: +/+, cre/+; fl/fl, +/+; +/+, +/+;
Conditional convert to Germline Knockout
Wildtype allele(WT)
WT-F WT-R
E1
E2
E3
E4
Targeted allele(fl-neo)
Optimizing Breeding Performance
Litter fostering
Determining pregnancy
Mating numerous females simultaneously
Managing Small Colonies
Maintain a minimum of six breeding pairs
Breeding Schemes
Choosing a Breeding Scheme
What genotypes do I need? most efficient way to produce them? Will appropriate controls be available? genetic background How are genotypes identified? phenotype? molecular assay (e.g. PCR)? What about Linkage? X-linked or multiple genes? Reproductive issues (sterility) Phenotype issues (lethality)
General principles for breeding
Fundamentals of Mouse Reproduction
Sexual maturity: 6~8 weeks
Reproductive life span: 7~8 months
Fertility
Gestation: 18~21 days Generation time: 12 weeks Litter size: 2~12 pups/litter Weaning age: 18~28 days
Disasters come in many forms
Cryopreservation
For colony “sleep” For insurance and peace of mind….development and basic phenotyping of a GM mouse can take 2-3 years and cost more than ￥500,000