Class Notes (1,000,000)
CA (610,000)
UTSC (30,000)
Lecture 5

BIOC14H3 Lecture Notes - Lecture 5: Homologous Recombination, Diphtheria Toxin, Neomycin


Department
Biological Sciences
Course Code
BIOC14H3
Professor
Patrick Mc Gowan
Lecture
5

This preview shows pages 1-2. to view the full 7 pages of the document.
1
BIOC14Winter2013 Lecture 5: Mouse Genetic Engineering and Genetic dissection of neural circuits (Article 1 and Article 2)
Article 1: Mouse Genetic Engineering
The mouse as a model organism
o Similar genome size, gene number, genome structure to humans most human genes have mouse counterparts
o Mutations causing diseases (or symptoms) in humans can cause similar disease (or symptoms) in mice
o Small, easy to maintain in the lab short breeding cycle (2 months), 5-9 pups/litter and approximately one liter per
month
o Transgenic approaches are now highly advanced
Generating Transgenic Mice (2 Approaches)
I. DNA microinjections into single cell embryos
o Produce transgenic mice containing multiple copies of randomly integrated DNA
DNA microinjection into single cell embryos
o 1. Fertilized once-cell eggs for DNA microinjection are obtained from superovulating female mice that have been
mated to stud made
o 2. Purified experimental DNA is directly injected into the male pronucleus of a fertilized mouse egg using a
microneedle male pronucleus is the nucleus of the sperm from the male (the sperm is in the egg, but transfer of
DNA is not yet complete)
o
o when multiple copies of DNA are introduced into nuclei, they are integrated into random sites within the mouse
genome
o DNA copies are integrated as head to tail concatemers due to homologous recombination copies all point in the
same direction (because they are lined up in homologous recombination in the same way and then pieces exchanged)
o Suggested that mammalian somatic cells possess enzymes for mediating somatic recombination at the time, they
did not think that somatic cells (cells that are not used in sexual reproduction) would contain machinery for
homologous recombination
o 3. Following microinjection, 20-30 eggs are impanted into the oviduct of pseudopregnant surrogate female (recently
mated to vasectomized male) female is bread by infertile male, such that all symptoms of pregnancy occur (and egg
without embryo develops)
o 4. After 19 days, the transgenic mice pups are born: transgenic litter mice are identified by PCR analysis of tail
samples performed at 3 weeks of age
Application of pronuclei injection methods
o transgenic mice are often generated to :
o (over)express wild type genes
o (over)express mutant genes
o (over)express reporter genes Gren fluorescent protein, Red fluorescent protein, LacZ
o
Cell type specific overexpression of a transgene
DNA microinjection into single cell embryos
2. Purified experimental DNA is directly injected into the male
pronucleus of a fertilized mouse egg using a microneedle
1. Fertilized one-cell eggs for DNA microinjection are obtained from
superovulating female mice that have been mated to a stud male
9.5 day transgenic embryos
wildtype and GFP
GFP transgenic mouse
Adult transgenic mouse expressing GFP

Only pages 1-2 are available for preview. Some parts have been intentionally blurred.

2
o
o cell-specific promoters can be used to direct expression of a gene to specific cell type in transgenic mice
o Pax6-GFP Pax6 is a "master control" gene for regulating eye, nose, central nervous system and pancreatic
development which is targeted to express the GFP
II. Use of homologous recombination at defined genomic loci
o Create gene knockouts (Kos) or replacements (Knock-ins)
o
o Positive selection: targeted gene is inactivated by inserting a marker gene that makes the cell resistant to antibiotics
(example: Neomycin)
o Homologous recombination is achieved in ES (embryonic stem) cells using electroporation
o
o Problems with homologous recombination unwanted random insertion is much more frequent than homologous
recombination events; having one seletion marker (Neo R) provides no selection against random insertions
o Solution: introducing a negative selection marker
o
Cell type specific overexpression of a transgene
Cell-specific promoters can be used to
direct expression of a gene to specific
cell type in transgenic mice.
Cell type specific promoter Gene of Interest
Cell type specific overexpression of a transgene
Cell-specific promoters can be used to
direct expression of a gene to specific
cell type in transgenic mice.
Cell type specific promoter Gene of Interest
Pax6-GFP
Generating transgenic mice
(By homologous recombination)
2 1 3
Neo R
+
Genome:
By using homology arms, one can
target any area of the mouse
genome
1 3 Neo R
Genome:
(targeted)
*** Positive selection: Targeted gene is inactivated by inserting a marker
gene that makes the cell resistant to antibiotics (e.g. Neomycin).
Targeting vector :
Generating transgenic mice
(By homologous recombination)
Homologous recombination is achieved in ES cells
using electroporation
Problems with homologous recombination
Unwanted random insertion is much more frequent than
homologous recombination events.
Having one selection marker (Neo R) provides no selection
against random insertions.
Solution: introducing a negative selection marker
2 1 3
Neo R
+
Genome:
Targeting vector : Diphtheria toxin
*** Diphtheria toxin: a negative selection cassette outside of the
homology arms
You're Reading a Preview

Unlock to view full version