Transcript Mouse-genetics-final-exam

Mouse Genetics
Final Exam Review
Why study vertebrates/ mice?
Reverse Genetics
• What is it?
– Starting with a specific genotype and figuring out
the phenotype
• Why is it used in mice?
– Reverse genetics is slow and expensive in mice
– Can test a candidate gene’s effects on complex
behaviors, organs, etc.
Tools for forward genetics in mice
1. Transgenes
2. Knock-ins
Transgenes
• What are they?
– Engineered pieces of DNA that have been inserted
randomly into the mouse genome by integration
• Why use transgenes?
– Any situation where you want to add in a gene
and not remove or replace anything.
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Test sufficiency/ complementation
Label cell types/ organs of interest
Add toxins
Promoter bashing
Etc.
What do you need to make a
transgene?
• A promoter- initiates transcription
• Your transgene- include a stop codon if
protein encoding (as opposed to RNAi)
• An intron- stops mRNA from being seen as
foreign by the cell
• Poly(A)- stop translation
How can you make the transgene
construct?
1. Bacterial plasmid
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pros: easy and cheap
Cons: small (20 kb)
can be subject to position effect
need to separate from vector
2. BAC (bacterial artificial chromosome)
-pros: bigger (100-150 kb)- therefore can include more
regulatory elements
-cons: can be subject to position effect
3. Lentivirus
-pros: less susceptible to position effect (targets active genes)
can be used for different species
no pronuclear injection
-cons: small
can disrupt essential genes
How do you introduce the construct
and make the mouse?
Using a plasmid or BAC:
1. Make the transgene cassette and separate if
from the vector DNA
2. Inject into male pronucleus of fertilized eggs
before the nuclei have fused
3. Transfer the transgenic eggs to a surrogate
mouse
4. Pups are born- genotype them to determine if
the transgene has integrated.
5. Mate the founders with WT mice to see if you
get transmission of your transgene.
How do you introduce the construct
and make the mouse?
Using a lentivirus:
• Same strategy as before, just no nuclear
injection.
• Incubate your fertilized egg with the viral
supernatant instead.
What problems may you encounter?
No expression or mis-expression of transgene.
• Position effect- transgene inserted into a part
of the genome not accessible by the
appropriate transcriptional machinery.
– Solution? Try using a lentivirus
• Your construct may have lacked the necessary
regulatory regions
– Use a BAC to get more upstream regulatory
sequence.
Gene Targeting
• What is it?
– Replacement or modification of an endogenous
gene with a sequence of interest
• Why use it?
– Knockouts
– Replacements
– Targeted mutations
What do you need to make a gene
targeting construct?
Components you must have:
– arms of homology to allow recombination into the endogenous locus- at least 4kb on
one side and 1.5kb on the other
– Neomycin (or other antibiotic) resistant marker with it’s own promotor (usually a
ubiquitous promoter), start and stop codons, and poly(A) tail- to select your ES cells
– Put Frt or LoxP sites around your antibiotic resistance marker so you can remove it later
Other tools you can use in gene
targeting constructs
• IRES (internal ribosomal entry sites) elements- allow you to
translate multiple proteins from the same mRNA (allows
7mG-cap independent translation)
• Reporter Genes (fluorescent proteins, LacZ, etc)- can be
used in two ways
– Fusion proteins- marks where IN THE CELL your protein of
interest is
– Cell labeling- marks WHICH CELLS are expressing your gene of
interest
• Use of tau:GFP fusions to label axons
• Toxins (such as diphtheria toxin)- to kill cells of choice
• Lox/ Cre and Flp/ Frt systems to selectively remove portions
of your construct
• Inducible promoters (tetracycline regulatory systems)- to
allow or inhibit expression of your targeting gene
How to make a construct for gene
targeting
1. Order a BAC clone spanning your region of
interest.
- This should be from the same strain. Why?
- So arms of homology do not contain SNPs
2. Screen a genomic library
3. PCR your region of interest
4. Synthesize it
How to make a “gene targeted” mouse
1. Add your construct into ES cells from male brown
mice and electroporate.
2. Select with appropriate antibiotics to get only cells
which have undergone homologous recombination.
3. Inject the ES cells into blastocysts from black mice.
4. Place blastocysts into recipient female. Chimeras are
born!
5. Mate chimeras to wild-type black mice
6. Look for brown progeny.
7. Mate to Flp/ Cre containing mice to remove selection
cassette
What do your chimeras look like?
What does this mean?
• All black pups
• Pups with variegated coats
• All brown females
• All brown males
What problems might you encounter?
• Aberrant expression of knock-in
– Forgot to “Flip-out” your antibotic resistance gene
– It has it’s own promotor and poly(A) tail, which
could lead to increased transcription and stability
of your knock-in construct
Other tools in mouse genetics
1. In-situ hybridization (ISH)
2. Nuclear cloning
In-situ hybridization
• Create a probe complementary
to your mRNA of interest. Label
this probe to determine where
your gene of interest is being
expressed.
Nuclear Cloning
• Creating an animal from a single adult cell
Notes about mice
• Sex- presence of Y chromosome determines
sex- XX= female, XY= male
• Because females have two X chromosomes,
the gene products must be equalized.
– X-inactivation- one X chromosome is stochastically
and irreversibly inactivated.
• If you have a loss of function mutation on the
X chromosome, here is what mutants will look
like:
– X(lof)/ X female=
– X(lof)/ Y male=