Coding region

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