Download Gene Targeting

Yeast as a Model
System II
MBIOS 520/420
October 4, 2005
MBios 420/520 Course Announcements
• Review Session Time?
• Sample Exam
• Study guide for exam will be provided next class
• Other issues/questions?
Yeast Homologous Recombination
• Homologous recombination (HR) is exchange or crossover
that occurs between two (nearly) identical DNA sequences
• In mammalian cells, this process occurs very rarely
• In yeast, HR occurs very frequently, due to small genome and
different recombination machinery
• This can be used to target or “tag” a gene with a marker, to
knockout a gene or to replace a gene with a mutated version
Marker or mutation
Gene X
Gene X
Plasmid
Homologous
Recombination
Chromosome
Gene X
Gene X
Homologous Recombination
Enzyme nicks
Strand
Exchange
Strands Join
A
B
Enzyme nicks
on axis A
Enzyme nicks
on axis B
Sister chromatids (dsDNA)
Homologous Recombination – Double Crossover
• If a double crossover event occurs, only DNA between the
two recombination sites is exchanged:
• We can introduce a plasmid into yeast and exchange can
occur:
Gene Targeting
• Homologous recombination can be used to “tag” a gene with
a marker in order to detect its inheritance
• For example  Let’s say we want to be able to detect the
presence of a specific allele of the gene YFG, which we will
call YFG*
• If YFG* has no easily measurable phenotype associated with
it, we can tag it with a marker that we can detect
• In our example, we will tag YFG* with a URA3 and transform
it into a yeast strain that can’t produce uracil
• If we linearize a plasmid that has URA3 and YFG*, the end
sequences will recombine with their identical counterpoint on
the yeast chromosome
Gene Targeting
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Gene Targeting – A Practical Example
• So we’ve tagged YFG* with a URA3 gene and inserted it into
one chromosomal copy in a URA3- mutant
• As an example, let’s say we suspect that YFG* causes
resistance to hygromycin and that yeast with YFG only is
susceptible to hygromycin
YFG* / YFG*
YFG*
From the gene targeting
on the previous slide we
already have a yeast of
the genotype YFG* / YFG.
YFG
w/o uracil
w/ hygroymicin
100% of
hygromycin
resistant yeast
have URA3. YFG*
must be a
hygromycin
resistance gene!
YFG* yeast also
have URA3. Let’s
replica plate on
media w/o
uracil.
YFG* / YFG
YFG / YFG
When this yeast
reproduces, three
genotypes will result.
Now let’s plate these
with hygromycin.
Creating Gene Knockouts by Transplacement
• We can use HR to create gene
knockouts by replacing a wild type
copy of a gene with a gene that has
an insertion
• Insertion is a selectable marker
gene so we can identify knockouts
• Problem: selectable markers are
dominant, so how do we get stable
knockouts that won’t segregate
Tetrad Analysis
• Yeast cells can be either haploid or diploid; when in the
diploid state they don’t mate
• Yeast can be induced to produce haploid spores under low
nutrient conditions
• By microdissection, we can separate the four haploid spores
(called a tetrad) and culture each one separately
• This allows us to isolate mutants that are hemizygous for a
given knockout or mutation
• If the knockout is lethal, half of the spores will not survive to
form colonies
Tetrad Analysis
Here we continue
the example of
the YFG gene
with the URA3
insert.
Our yeast was
heterozygous, but
if we isolate
spores we can
get a hemizygous
mutant.
If the YFG gene is
essential, all yeast with
URA3 will not survive.
Studying Higher Eukaryotic Genes in Yeast
• Commonly conserved genes, like cell cycle genes, are so
similar between yeast and mammals, that they can be
switched
• This allows us to study the function of mammalian genes
without having to use a mammalian system
• Ex: If we wanted to study the DNA binding domain of a human
transcription factor, we could mutant it, tag it with a marker
and replace a similar yeast gene with it
• A technique called plasmid shuffle can be used to replace
essential genes in yeast with their mammalian counterparts
Plasmid Shuffle
We’ve created a yeast
knockout of TPK1 gene.
Because TPK1 is essential,
yeast needs a plasmid
with TPK1 to stay alive.
Transform with mouse TPK
gene in another plasmid.
Let these yeast cells lose
one plasmid by removing
selection criteria.
Plate on media to show
that one kinase gene is
active.
Replica plate w/o leu2 to
show that mouse TPK
gene is in yeast.
TPK
homolog