Download Transgenic Animal Models

Transgenic Mouse: Generic term for an engineered mouse that has a normal
DNA sequence for a gene replaced by an engineered sequence or a
sequence from another organism.
Knockout Mouse: A transgenic mouse in which the normal gene is missing or
engineered so that is not transcribed or translated. “Knocks out” that gene.
Knockin Mouse: A transgenic mouse in which the engineered “transgene” is
subtly manipulated to: (A) alter the function of the gene (e.g., replace one
amino acid with another in a site to determine if that site is essential for the
protein’s function); (B) change transcription rate to overproduce or
underproduce the gene product; or (C) create a fluorescent gene product to
map its distribution in tissue.
Conditional Knockout (Knockin) Mouse: A transgenic mouse in which the
transgene is knocked out (or in) in specific tissues, at a specific
developmental stage, or in response to an exogenous substance (e.g., an
antibiotic).
Transgenic Organisms
General Outline:
• Infect blastocyst cells/sperm with viral vector with the gene
of interest.
• Hope that in some cells homologous recombination will
insert the DNA section of interest into the target cell’s
chromosome.
•Select chimeric organisms.
•Breed until the transformed DNA is found in a germ line.
(1) Get the nucleotide sequence of the gene of interest. Including upstream
and downstream nucleotides.
agctta
tcgaat
Gene of Interest
cgatc
gctag
Downstream
DNA (unique)
to the gene,
usually > 1 kb
Upstream
DNA (unique
to the gene),
usually > 1kb
(2) Construct the desired DNA sequence (i.e., the transgene), adding
a gene for antibiotic resistance, but keeping the upstream and the
downstream nucleotides.
agctta
tcgaat
Desired Gene
Antibiotic Resistance Gene
cgatc
gctag
(3) Micropipette embryonic stem cells from the inner cell mass of a
blastocyst (i.e. early mouse embryo) in a strain with a physically recognizable
phenotype (e.g., pigmented).
(4) Culture the cells with many copies of the manufactured transgenic
DNA complex. Short bursts of an electrical current allow the DNA to pass
through the plasma membrane into the cell (electroporation).
(5) Cells will divide in culture and some of them will incorporate the transgenic DNA
strand into the chromosome (homologous recombination). After a sufficient number
of cell divisions, add the antibiotic. This will preferentially kill those stem cells that
have not incorporated the transgenic strand (black dots), giving a good harvest of
those that have incorporated the strand (red dots).
+ antibiotic
(6) Insert the stem cells into the blatocyst of a mouse with a different genetic
background trait (e.g., an albino if the original stem cells came from a
pigmented mouse).
(7) Implant the new blastocysts into a pseudopregnant female with a visible
phenotype different from the blastocyst phenotype (e.g., albino if the
blastocyst is pigmented).
(8) Offspring that have pigmented sections are chimeras that have incorporated
the transgenic sequence into their cell lines. Select them for further breeding.
(9) Keep breeding the offspring of the chimeras until some fully pigmented mice
are born. A fully pigmented mouse means that the transgenic germline generated
one of the gametes that resulted in that mouse. Genotype the mouse to determine
the genotype at the desired locus and the insertion point(s). (Most will be
heterozygotes for the wild type allele and the transgenic allele).
(10) Mate two heterozygotes and genotype their offspring. This will give all three
genotypes--wild type homozygotes, heterozygotes, and transgenic homozygotes.
(11) Compare the three genotypes on the phenotype of interest.
(12) Problems:
(a)
(b)
(c)
(d)
(e)
Multiple insertions: too much protein.
Insertion into a life-necessary gene: lethality.
Insertion into a gene leading to gene-silencing: no protein.
Insertion in a different area can lead to differential gene regulation.
Background genotype can be limiting.
Conditional Gene Expression
Generate a transgenic line. Engineer the transgene section so that
it contains unique sections (a loxP site) to a specific Cre
recombinase (enzyme that catalyzes recombination) at the
sites.
Mate this line with the desired Cre recombinase mouse strain.
E.g., one that has the Cre recombinase in the hippocampus.
There is a very large number of Cre recombinase mouse
lines, each with tissue-specific, developmentally specific, or
exogenous chemical specific properties (e.g., responds to
tetracycline).
The offspring will then have the gene knocked out in the desired
tissue, at the desired developmental stage, or when the
chemical inducer is administered. The knockout occurs when
cells divide and the transgene “loops” during recombination
and is not included in the recombinant chromosome.
NOTE: This is only important in cells that are actively dividing.
Hence, it is not suitable for most neurons.
Gene “Therapy”
(gene transfer)
(1) Insert the gene of interest into a benign viral vector. (Choice of
vector can be important).
(2) Use microinjection to flood a specific area/nucleus of the
brain with the viral vector.
(3) Some neurons will take in the virus and incorporate the
transgene into the nuclear DNA.
(4) Afterward, assay the area for mRNA activity, proteins, etc. to
see the effect of the transgene.
Gene Therapy
Notes:
Often a marker gene is also included in the viral vector to
assess how well the neurons take up the vector (e.g., GFP or
green fluorescent protein).
Useful to study overexpression of a gene product. (E.g.,
overexpression of CREB in hippocampus).