Transgenic animal approaches

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Transgenic animal approaches a. Use of
transgenes to study gene function in
non-vertebrate model organisms continued Gene
targeting in Drosophila by homologous
recombination -in addition to allowing one to
express a gene in a particular spatial and
temporal pattern, another major use of transgenes
is as a tool to knock out the function of a
particular gene -as we have seen, RNAi
approaches can be used to inhibit gene function
but to get a stable knock-out we need to go in
and disrupt the gene in the chromosome -the key
to targeted gene knock-outs is using homologous
recombination to recombine an engineered
transgene with its endogenous counterpart, thus
permanently disrupting it -in model organisms
such as yeast and mice, introduced DNA readily
recombines with homologous sequences in the
chromosome but this is not the case in
Drosophila -a method has recently been developed
that prompts Drosophilas recombination and
repair machinery to integrate DNA at a target
locus by homologous recombination
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-the method relies on three components (i) a
transgene that expresses a site-specific
recombinase (ii) a transgene that expresses a
site-specific endonuclease (iii) a transgenic
donor construct that carries recognition sites
for both the above enzymes and DNA from the
locus to be targeted -flies with all three
components are generated through simple genetic
crosses
-as we saw with homologous recombinations between
the pBacPAK8/9 transfer vector and the BacPAK6
baculovirus, the frequency of recombination is
greatly increased by having double-strand breaks
in one of the DNAs -the overall principle of the
technique is to excise the donor transgene from
the chromosome in a circular form, introduce a
double-strand DNA break into the transgene and
hope that it recombines with the endogenous
target gene in the chromosome
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-some defects are introduced into the transgene,
such that the final tandem duplicated genes that
result in the chromosome are both
nonfunctional -in order to get the donor
transgene to excise from the chromosome in a
circular form, the donor transgene is
constructed with flanking target sites (FRTs)
for the FLP site-specific recombinase of
yeast -a marker gene such as white is also
included in the construct -when FRTs are placed
in the same orientation (i .e. as direct
repeats), the FLP recombinase directs
recombination between these and the intervening
DNA is released as a circle
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-the Flp recombinase is supplied by a heat
shock-inducible Flp transgene on another
chromosome -in order to introduce a
double-stranded break in the donor transgene, the
18bp recognition site for the yeast I-SceI
endonuclease is inserted into the donor
transgene -this 18bp sequence is not found
anywhere in the Drosophila genome, therefore
I-SceI will only cut the donor transgene
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-the I-SceI enzyme is supplied by a heat shock
inducible transgene on the same chromosome as the
Flp transgene -in order to do a recombination,
flies bearing the donor transgene are crossed to
flies bearing the heat shock inducible I-SceI
and Flp transgenes -progeny flies will contain
all three components and when exposed to elevated
temperature the donor transgene is released as a
circle and cut -the donor transgene is then free
to recombine with the target gene -when this
event occurs in the germline, one will be able to
pick up progeny with the target gene knocked out
in every cell -the system has been tested by
knocking out the pugilist gene, null alleles of
which show eye colour defects (pugilist encodes
enzyme methylenetetrahydrofolate dehydrogenase,
MTH) -various means were used to determine that
the pugilist gene had been knocked out
by homologous recombination with the transgene
(i) appearance of the pugilist null
phenotype (ii) genomic Southern
blotting (iii) chromosomal in situ
hybridization with a probe for the
white marker gene
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Introducing genes into C. elegans -rather than
relying on transposon- mediated incorporation
into the chromosome, transgenes are introduced
into C. elegans as simple DNA samples which end
up being maintained in the worm as heritable
extrachromosomal arrays -in early attempts to
make transgenic worms, DNA was injected directly
into the nuclei of oocytes, which lead
to integration into the genome at relatively high
frequency -injections directly into the nucleus
are technically demanding , and in the more
popular approach, DNA is injected into the
cytoplasm of the mitotically active, syncytial
gonad of the hermaphrodite worm -the injected
DNA can be stably passed through the germline as
extracellular arrays- C.elegans will replicate
any DNA introduced into the germline and no
special vectors are needed
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-DNA is injected with a marker DNA conferring a
visible phenotype, so that transformant worms
can be readily identified (equivalent to white
gene in Drosophila transformation
vectors) Commonly used markers -lacZ reporter
gene -GFP reporter gene (transformants can
be identified as live worms with a fluorescence
dissecting scope) -various genes conveying a
phenotype e.g. inject pha-1(e2123ts) mutant
worms with gene of interest and wild-type pha-1
gene. The nontransformant worms will die at
25oC, but transformants will survive e.g.
inject dominant mutant collagen
gene rol-6(su1006), which causes worms to
roll and move in circles, a phenotype easily
seen with a dissecting scope
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-much of the injected DNA is only transiently
expressed in the F1 and is not inherited by the
F2 -extrachromosomal arrays are the predominant
form of injected DNA that is transmitted to the
F2 -array-containing lines are derived by
selecting F1 progeny with the marker phenotype
and then checking their progeny for the
marker -the formation of arrays is favoured
by high DNA concentrations in the injection
solution (gt100µg/ml) -arrays bigger than about
700 kb become heritable as extrachromosomal
elements -these behave like fragments of
endogenous worm chromosomes with an average 50
inheritance in each generation -each array can
contain hundreds of linked copies of the
injected DNA molecules
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-extrachromosomal arrays can be stabilized by
gamma-ray or X-ray induced integration into a
chromosome -the mechanism of this integration
is unknown, but presumably involves breaks
induced by irradiation -a transgene expression
system akin to the Drosophila GAL4-UAS system has
not been developed for worms,however, vectors
have been developed into which one inserts cloned
promoters and a gene of interest (this is like
original Drosophila transformation vectors) -an
example of such a vector is pPD49.26 which
contains -a synthetic intron and the 3 region
from the unc-54 gene -three MCS (polylinker)
regions for inserting (I) promoter/enhancer
(ii) gene of interest (iii) downstream
regulatory sequences -vectors bearing a variety
of interesting tissue-specific and inducible
promoters are available -as with Drosophila,
homologous recombination between transgenes and
their endogenous counter- parts occurs rarely in
C. elegans and at this point in time transgenes
do not offer a convenient way to knock out gene
function in worms -RNAi is the most heavily used
approach at the moment to achieve directed
disruption of gene function in worms
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Use of transgenes to study gene function in
mice Creating transgenic mice by microinjection
of fertilized oocytes -for the past 20 years,
researchers have been able to introduce DNA into
mice by microinjection into fertilized eggs -in
order to get large numbers of eggs for
injection, superovulation is induced in females
by injecting them with pregnant mare serum
gonadotrophin (PMSG) and human chorionic
gonadotrophin (hCG) -superovulating females are
then mated, sacrificed, and oocytes
removed -DNA is injected into the male
pronucleus of oocytes using a glass microneedle
controlled by a micromanipulator -the oocyte is
held in place by a pipette under negative
pressure -about a picoliter of buffer
containing several hundred molecules
of linearized DNA is injected
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-the injected DNA generally occurs randomly at a
single site, the site of insertion possibly being
a double-stranded break occurring during
injection -for most transgenes, 1- 50 copies are
integrated, although up to a 1000 copies have
been seen -transgenes usually occur as a tandem
array of head-to-tail fusions -injected eggs are
implanted into a pseudopregnant mouse
(previously mated with a vasectomized male) and
the progeny mice checked for the presence of the
transgene -this is done by taking tail tips or
ear punchs and checking them by PCR or Southern
Blot -as transgene integration often occurs
after the one-cell stage, the first generation
mice may be mosaic for the transgene and may not
contain it in the germline -mice that score
positive by PCR or Southern are backcrossed to
nontransgenic mates to identify founder animals
with germline integration of the transgene
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-transgenes introduced into mice by oocyte
injection can be used for various purposes, for
example in rescue experiments to prove that a
mutant phenotype is caused by loss of the
endogenous gene -such rescue experiments usually
require expression of the gene under control of
its own promoter/ enhancer sequences -as
regulatory domains may be several hundred
kilobases long, one may have to inject large
stretches of genomic DNA which can be
accommodated in vectors such as bacterial
artificial chromosomes (BACs) or yeast
artificial chromosomes (YACs) -similar to what
we saw with flies and worms, one can express
mouse transgenes in particular tissues using
well characterized promoter/enhancer sequences
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-transgenes are essential for characterizing the
complex regulatory regions of mammalian
genes -for example, one can dissect the enhancer
region of a gene by fusing fragments of it with a
lacZ reporter gene bearing a minimal promoter and
seeing what patterns b-galactosidase is expressed
in in transgenic mice