1
Coding region
ATG: Translation start Translation stop
Transcription start (mRNA start) (mRNA end) poly-adenylation
exon intron
Mature mRNA
Genestructure
AAA
cap 5’UTR 3’UTR polyA tail
Regulatory regions
2
Gene of interestHeterologous regulatory region
Heterologous regulatory region
cDNA from gene of interest
Transgenic gain-of-function (transgene injection in fertilized eggs)
Gene of interest with it’s own regulatory elements
3
BAC transgenics
~ 200 kb
Regulatory elements can many kb away from gene
GFP recombineering
4
NEO
Gene inactivation (constitutive) by gene targeting in ES cells
Replace critical exon with Neomycin resistance gene
NEO Simple targeting construct
Heterozygote +/-
NEO
NEO
Homozygote -/-
Positive selection
5
NEO
Targeted gene knock-in (knock-out) in ES cells
NEOCRE
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.
Targeting construct
NEONEOCRE
NEONEOCRE TKDTa
Positive selection
Negative selection
Note insertion between transcription and translation start sites
6
CRE
ires-Cre
Gene of interest
NEONEO
Frt sites
Use of ires-Cre does NOT disrupt function of gene of interest
CRE
Gene of interest after FLP recombinase action
7
Mouse with Cre expressed from gene of interest, e.g. Agrp-Cre
a) Breed with mouse containing conditional allele of gene of interest to either inactivate (or activate) that conditional allele
b) Inject a Cre-dependent virus (to achieve brain region specificity)
C -------------N
ITR promoter gene of interest WPRE pA ITRbackwards
N -------------C
ITR promoter gene of interest WPRE pA ITRbackwards
DIOFLEX
Double recombination results in expression of gene of interest
N -------------C
First recombination (inversion), intermediate stage
Second recombination (deletion), locks it into inverted position
8
NEO
Conditional gene inactivation
loxP sites Frt-NEO
Targeted allele
Remove Frt-NEOWith FLIP recombinase
Neo may disrupt function = KO
Cell-specific expression of Cre recombinase (transgene or knock-in)
CRE
Breed to homozygosity and introduce Cre gene
Removed with FLPrecombinase
9
Cre-mediated recombination wherever Cre expressed (loss of critical exon)
CRE
CRE ERt
Cell-specific control temporal control with tamoxifen
Cre-mediated recombination wherever & whenever Cre is expressed (loss of critical exon)
10
NEO
Conditional gene activation
loxP sites
Inactive allele
CRE
loxP site
Active allele
Cell-specific promoter
11
NEO
Conditional gene activation
loxP sites
Inactive allele
CRE
loxP site
Active allele
•Reporter gene•Ion channel•GPCR•Toxin
Cell-specific promoter
12
NEO
loxP sites
Inactive allele
NEO
Targeting constructmutation
CRE
loxP site
Active allele with mutation
Targeted introduction of a mutation
mutation
13
Conditional exon swap (double recombination)
NEO
Targeting construct
Remove frtNeo with FLP recombinase
Double recombination with Cre recombinase
Frt siteLox P siteLox P site (different)
a
a
b
b
2-step recombination only between like lox P sites following
path a or b
