Es cells with the mutant gene into a wild type embryo

ES cells with the
mutant gene into a
wild-type embryo
Step 6: Implanting the
embryo into
“pseudopregnant”
mouse
http://www.ncbi.nlm.nih.gov/
books/NBK26818/figure/A1650/?
report=objectonly

Ques%ons?

Problems with genes that act in multiple
tissues, at multiple times
How to activate the mutation at a time or in the
tissue of choice?
Solution: “conditional” knock-out, using microbial
recombinase systems:
Cre recombinase, recognises loxP sequences
(originally found in P1 bacteriophage)
FLP recombinase, recognizes FRTsequences (from
yeast)

Tissue-specific disruption of a gene using Cre/
LoxP system
floxed gene “deleter” mouse, expresses cre
recombinase:
- Depending on the promoter to
which Cre is fused, it can be
expressed either in all cells, or just
in a subset of cells (brain, liver, etc)
For more information:
http://cre.jax.org/introduction.html

Tissue-specific disruption of a gene using Cre/LoxP
system

At home: summarize the differences between
knockout, knock-in, and conditional knockout
Knockout Knock-in Conditional
knockout
Purpose
Position of neoR
(intron? exon?)
Position of
regions of
homology
Position of TK
Position of loxP

In Achondroplasia paper, the researchers flanked
(“floxed”) neo gene with loxP sites
In tube
In
animal
In
animal
Cre recombinase deleted neo gene

Generating a mouse model of Achondroplasia:
First, the targeting vector is generated
In tube
In ES
cells
In
animal

Summary