Why and How to make a Gene Knock-out mutation in Mice

1
Why and How to make a Gene Knock-out mutation in
Mice

• Why would you want to do this?
• Making the knock-out construct
• Engineering mouse ES cells heterozygous for the
  knock-out mutation
• Engineering chimeric mice with germ cells
  heterozygous for the knock-out mutations
• Creating mice M/m and m/m to study their phenotype

2
1. Why make a Gene Knock-out in Mice? An example

• You are studying human disease M-Disease
• Rare but devastating autosomal recessive
• You clone M-Gene which you think is the gene
  mutated in M-Disease
• You can prove this by making a mouse M-gene
  mutant and showing it gets M-Disease

3
2. Making Gene Knock-out Construct

• Clone mouse M-Gene
• show mice have M-Gene
• By southern blot analysis
• Mouse genomic DNA cut with REs
• Human M-Gene cDNA as probe
• Pattern should be as expected for a single M-Gene

4
2. Making Gene Knock-out Construct

• Clone mouse M-Gene
• show mice have M-Gene
• Find M-Gene clone in mouse genomic library
• Buy lambda phage mouse library
• Probe with human M-Gene cDNA
• Identify M-Gene clone
• Confirm restriction map mouse M-Gene clone

5
2. Making Gene Knock-out Construct

• Clone mouse M-Gene
• Clone the neoR gene into exon 2 of M-Gene
• Use HindIII fragment containing M-Gene
• Clone neoR gene into EcoRI site in exon 2

6
2. Making Gene Knock-out Construct

• Clone mouse M-Gene
• Clone the neoR gene into exon 2 of M-Gene
• Clone the thymidine kinase gene (tk) onto end
• Use the HindIII fragment containing mutant M-Gene
• Clone the tk gene into the BamHI site

7
3. Engineering Gene Knock-out ES cells

• Use ES cells from Brown (A/A) mouse
• A/A brown A/a agouti a/a black
• Inject knock-out construct into ES cells
• Select transformants in growth medium containing
  G418 Ganciclovir
• Only colonies that grow gene replacements
• A/A M/m
• Confirm M/m genotype by southern blot (HindIII
  digest)

8
4. Engineer chimeric mice with Germ line
Knock-out ES cells

• Inject ES cells (A/A M/m) into a/a M/M early
  embryo
• Implant embryo into a/a M/M surrogate mother
• Examine litter for mice with a mixture of brown
  and black fur
• Chimeric mice some cells a/a M/M some cells A/A
  M/m

9
5. Create M/m and m/m Mice

• Cross chimeric mice with a/a M/M mice
• If A/a (agouti) pups are born, A/A M/m cells were
  in the chimeras germ line
• Other possible pup genotypes (A/a M/M a/a M/M)
• Check A/a mice for M/m genotype by southern blot
  (HindIII digest)
• Cross two M/m mice and look for m/m pups by
  southern blot
• If no m/m mice are found
• If healthy m/m mice are found
• If m/m mice that develop M-Disease are found