Coding region

1
Transcription start (mRNA start) (mRNA end)
poly-adenylation
ATG Translation start Translation stop
Gene structure
exon intron
Regulatory regions
Coding region
Mature mRNA
AAA
cap
5UTR 3UTR polyA tail
2
Transgenic gain-of-function (transgene injection
in fertilized eggs)
Gene of interest
Heterologous regulatory region
cDNA from gene of interest
Heterologous regulatory region
3
BAC transgenics
GFP
recombineering
200 kb
Regulatory elements can many kb away from gene
4
Gene inactivation (constitutive) by gene
targeting in ES cells
Simple targeting construct
NEO
Replace critical exon with Neomycin resistance
gene
NEO
Heterozygote /-
NEO
Homozygote -/-
NEO
5
Targeted gene knock-in (knock-out) in ES cells
CRE
Targeting construct
NEO
NEO
Cre (or a reporter gene) is now driven by
regulatory elements of gene of interest.
Critical parts of gene of interest are removed so
it is also a null allele.
CRE
NEO
NEO
6
Conditional gene inactivation
Neo may disrupt function KO
Targeted allele
Frt-NEO
loxP sites
Remove Frt-NEO With FLIP recombinase
Breed to homozygosity and introduce Cre gene
CRE
Cell-specific expression of Cre recombinase
(transgene or knock-in)
7
Cre-mediated recombination wherever Cre expressed
(loss of critical exon)
CRE
Cre-mediated recombination wherever whenever
Cre is expressed (loss of critical exon)
CRE ERt
Cell-specific control temporal control
with tamoxifen
8
Conditional gene activation
Inactive allele
loxP sites
CRE
Cell-specific promoter
Active allele
loxP site
9
Conditional gene activation
Inactive allele
loxP sites
CRE
Cell-specific promoter
Active allele
loxP site

• Reporter gene
• Ion channel
• GPCR
• Toxin

10
Targeted introduction of a mutation
mutation
Targeting construct
Inactive allele
loxP sites
CRE
mutation
Active allele with mutation
loxP site