Article 1.

Mouse Genetic Engineering

Gene targeting in mice: functional analysis of the mammalian

genome for the twenty-first century
PMID: 15931173

material that is in the article that is talked about in lecture is testable

 The mouse as model organism
•  Similar genome size, gene number, genome structure
to human
- Most human genes have mouse counterparts

•  Mutations causing diseases (or symptoms) in human

can cause similar diseases (or symptoms) in mice
- sometimes difficult when comparing with behaviour

•  Small, easy to maintain in the lab

- Short breeding cycle (2 months)
- 5 to 9 offspring per litter and approximately one litter
per month

•  Transgenic approaches are now highly advanced.

 Generating transgenic mice
(two approaches)

1.  DNA microinjection into single cell embryos

: produce transgenic mice containing multiple copies of randomly
integrated DNA
randomly asserted

2.  Use of homologous recombination at defined genomic

loci
: create gene knockouts (KOs) or replacements (Knock-in)
DNA microinjection into single cell embryos
1. Fertilized one-cell eggs for DNA microinjection are obtained from
superovulating female mice that have been mated to a ‘stud’ male

_____trophins

2. Purified experimental DNA is directly injected into the male

pronucleus of a fertilized mouse egg using a microneedle
usually done with the male
nucleus, slightly bigger
 DNA microinjection into single cell embryos
•  When multiple copies of DNA are introduced into nuclei, they are
integrated into random sites within the mouse genome.

•  DNA copies are integrated as head to tail concatemers due to

homologous recombination. copies that follow each other

•  Suggested that mammalian somatic cells

possess enzymes for mediating somatic
recombination.

somatic cell stil has the embryonic machineary to mediate homologous recombination
 DNA microinjection into single cell embryos
3. Following microinjection, 20-30 eggs are implanted into the
oviduct of a pseudopregnant surrogate female (recently mated
to a vasectomized male)
female displaying all characteristics except not pregnant (mate with visectimized male)

19 days after implantation

2. After 19 days, the transgenic mice pups are born.

: Transgenic littermates are identified by PCR analysis of tail samples
performed at three weeks of age

http://www.youtube.com/watch?v=2KoLnIwoZKU

some may or may not have the foreign DNA

DNA microinjection into single cell embryos

http://www.youtube.com/watch?v=h-Bfc1GPWpE
Application of pronuclei injection methods

Transgenic mice are often generated to:

•  (Over)express wild type genes
unmodified gene

•  (Over)express mutant genes

•  (Over)express reporter genes (GFP, RFP, LacZ)

fluorescent genes
 GFP transgenic mouse
9.5 day transgenic embryos –
wildtype and GFP

Adult transgenic mouse expressing GFP

Cell type specific overexpression of a transgene

Cell type specific promoter Gene of Interest

link a cell type specific promoter to the gene of interest

Cell-specific promoters can be used to

direct expression of a gene to specific
cell type in transgenic mice.
Cell type specific overexpression of a transgene

Cell type specific promoter Gene of Interest

Cell-specific promoters can be used to

direct expression of a gene to specific
cell type in transgenic mice.

Pax6-GFP
 Generate transgenic mice
(two approaches)

1.  DNA microinjection into single cell embryos

: produce transgenic mice containing multiple copies of randomly
integrated DNA

2.  Use of homologous recombination at defined genomic

loci
: create gene knockouts (KOs) or replacements (Knock-in)
 Generating transgenic mice
(By homologous recombination)

Genome: 1 2 3

+ sequences of dna that you insert that flank you gene

of interest. specifically target a part of the genome
Targeting vector : Neo R

By using homology arms, one can

target any area of the mouse
genome

Genome: 1 Neo R 3
(targeted)

*** Positive selection: Targeted gene is inactivated by inserting a marker

gene that makes the cell resistant to antibiotics (e.g. Neomycin).
 Generating transgenic mice
(By homologous recombination)

Homologous recombination is achieved in ES cells

using electroporation
creates pores in the membrane that allow the DNA to enter

inactivated chemically

embryonic stem cell are pluripotent - can become any cell

 Mouse ES cells growing on fibroblasts

feeder cell layer - there to promote growth of Embryonic cells

 Problems with homologous recombination
- into locations that are not of interest
•  Unwanted random insertion is much more frequent than
homologous recombination events.

•  Having one selection marker (Neo R) provides no selection

against random insertions.
neomycin promotes positive selection - all cell with neomycin resistant gene (Neo R) will survive and proliferate while
others without it will die

Solution: introducing a negative selection marker

Genome: 1 2 3

+
Targeting vector : Neo R Diphtheria toxin

*** Diphtheria toxin: a negative selection cassette outside of the

homology arms
only ones that survive are the one that are inserted into the correct location
 What can happen in ES cells after
electroporation?
1.  No homologous recombination / no random insertion

2.  Random insertion

3.  Homologous recombination / no random insertion

-cell selecting for using this technique

If we grow the electroporated ES cells in neomycin containing medium….

Genome: 1 2 3

+
Targeting vector : Neo R Diphtheria toxin
chimeric - contains artificial DNA mixed
with original DNA
ES cell microinjection into a mouse blastocyst

http://www.youtube.com/watch?v=1m9kQuKXxEA
Applications of homologous recombination

•  Knock-in : targeted insertion of the transgene at a

selected locus in a chromosome. choose where you want it (multiple locations of body,
and not brain)
-  More consistent level of expression than the
pronuclei injection approach
-  More time consuming (vector assembly, identify
ES cells that have undergone recombination)

•  Knock-out : targeted deletion of a gene.

-  While pronuclei injection method and knock-in

method are typically used to express a certain
gene, knock-out is used to eliminate a gene.
 Applications of homologous recombination

•  (Conventional) knock-out approach:

-  Delete both alleles so that the gene is entirely absent

from all cells.

•  Conditional knock-out approach

-  Delete a gene in a particular organ, cell type, or at a

specific time point (can avoid compensatory effects).
by using a specific promoter

-  Circumvent lethality (~15% of knock-outs are lethal)

have to link it to a specific promoter in specific location as to not kill the cell

-  More powerful in defining gene function in physiology

and behavior
•  Conditional knock-out approach
: There are several different ways of making conditional knock-
outs. However, the most widely used method is the Cre-LoxP system
cre-recombinase
they can orient the DNA
 Conditional knock-out approach

genome: Gene of interest

Cre
If CRE recombinase is expressed…

…the gene between loxP sites is removed.

•  Conditional knock-out approach requires two components:

1.  Gene flanked by two loxP sites (Floxed mouse)

2.  Tissue specific Cre recombinase (Cre mouse)

only in specific location that you are interested in
 Floxed mouse: First step involves homologous
recombination
Genome: 1 2 3

+
Targeting vector : 2 Neo R

By using homology arms, one can

target any area of the mouse
genome

Genome: 1 2 Neo R 3
(targeted)

** Gene flanked by loxP sites (floxed)

Floxed mouse: Second step involves developing a
Cre mouse – typically done by pronuclei injection
Cre will only be expressed when the specific promoter expresses it?

Cell type specific promoter Cre

Together, this is an example of an ‘intersectional method’

* There are more than 1000 Cre mice available.
 Conditional knock-out by Cre-loxP system

- glutamate expression removed

bright = camp kinase

is expressed
 Genetic modification of behaviour:
What’s wrong with this transgenic mouse?

fear was taken out

http://www.youtube.com/watch?v=UJP8HKDfB7c

Nature 450, 503-508 (22 November 2007)

 What’s wrong with this transgenic mouse?

deletion in olfactory
bulb.
omix genes - cells that
that are omix positive die

specific to odors of
predators

http://www.nature.com/nature/journal/v450/n7169/suppinfo/
nature06281.html
Nature 450, 503-508 (22 November 2007)