Mouse Genetics Flashcards
what are some targeted approaches to creating mutant mice?
- CRISPR
- transgenic mice
- knockout mice
- conditional knockout mice
- knockin mice
- inducible gene expression
random mutagenesis
feed the mice a chemical mutagen like ENU and look for novel phenotypes
making transgenic mice
put a gene together and express that in a mouse by injecting the embryo with naked DNA (goes in randomly and can form concatemers)
mouse development
-when sperm meets the egg and you have implantation, form zygote within 12 hours
-embryonic day 4.5 we have late blastocyst with inner cell mass
-from the inner cell mass, we derive the embryonic stem cells (ESCs)
-these ESCs are used for gene targeting by homologous recombination
construction of the targeting vector
sequence of interest on either side of HR arms that direct where DNA insertion site (changes the target gene) is and allows for positive/negative selection using neomycin cassette
ES cell transfection
-cellular machinery for HR allows the targeting vector to find and recombine with target gene
-use HSV-tk on the vector so that you can ID if the vector has gone through HR or not when exposing the cells to an antiviral drug
proliferation of targeted ES cells
selection for presence of neomycin resistance cassette and absence of HSV-tk
injection of ES cells into blastocysts
-targeted ES cells are injected into blastocyst –> they mix and form a mosaic with the cells of the inner cell mass from which the embryo develops –> the injected blastocysts are implanted into a surrogate mother where they develop mosaic embryos
-in diagram: yellow cells of recipient blastocyst can be female (XX) or male (XY) and if the injected gene-targeted XY ESCs (red) go on to incorporate into what will become the germline, the mouse will turn male, regardless of the karyotype of the somatic cells
what are some limitations of blastocyst injection?
- obtaining chimeras- mutation may not allow for mouse development
- chimeras going to germline- the injected ES cells may not contribute to sperm development
- strain choice- some strains like the most common one used do not yield a lot of chimeras
- maintenance of normal chromosome numbers- mES cell lines are prone to chromosome loss/gain
- time and money
making mice with CRISPR-Cas9
when you create a double stranded break in DNA in sequence manner with added homology vector with fairly small homology domains, you can incorporate single stranded oligonucleotide and repair vector
what are the steps to making mice with CRISPR-Cas9?
- superovulation and breed
- isolate single cell zygotes and microinject with Cas9 components (Cas9 protein, CRISPR RNA, tracrRNA, single-stranded oligodeoxynucleotides) then culture to 2-cell stage embryo
- transfer 2-cell stage embryo to pseudo-pregnant recipient
- genotype pups to ID desired alleles
advantages to CRISPR-Cas9
don’t need ESCs, don’t have to worry about getting chimerisms, germline competency, and it’s good if you want to make small changes to the genome
Cre recombinase
-we have an exon we’re interested in and put in loxP sites that are recognized by a Cre recombinase, which we can express in trans
-Cre recombinase encoded in genome within a virus, within a protein, within an RNA –> as long as we can get protein to be expressed in the cell we are interested in and it will recombine 34 bp sequences (loxP sites) and form mini circle that cell excludes
-there are different types of lox sites: we focused on loxP sites that are 34 bp and if you put two heterotypic lox sites together, you won’t get Cre activity
what happens if loxP sites are in the same direction and bred to Cre?
everything within the loxP sites will be excised and you are left with one loxP site
what happens if the loxP sites are in opposite directions?
everything within the loxP sites will be inverted and you maintain both loxP sites
what are the options for transgenics?
- targeted integration with a specific locus- HR/CRISPR
- direct injection of linear DNA- happens with random insertion and often forms concatemers
- BAC transgenics- contain large regulatory elements and also is random insertion
- viral/retrotransposon- do this with a lentivirus and allows for insertion of single copy of mutation
how would you design a mouse that permanently inverts exon 2 of the fuzzy gene in the retina?
-put two heterotypic lox sites facing opposite directions
-apply Cre that works for two of the sites and you get inversion of the exon along with the one lox site moving to the other side of the exon
-apply Cre again to remove the lox site that is between the other two lox sites that are the same color
Cre expression
-transgenic with a specific promoter
-BAC expression
-knockin to a gene of interest
-temporal expression: CreERT2-tamoxifen regulated and tetracycline responsive Cre
Cre Knockout First Allele
-begin with tm1a allele that has the lacZ reporter allele that is flanked by one FRT site and one loxP site then there is the neomycin resistance cassette that is flanked by the loxP site and another FRT site then you have exon 2 flanked between two loxP sites
-if you want to create a mutant that has the reporter allele and null expression of the gene, breed to Cre first then you get removal of everything between the lox sites and you are left with one FRT site and one loxP site
-if you want to create a mutant that has the gene expressed, breed to Flp and you are left with one FRT site and then a floxed exon
what is a knockout first allele?
tm1a
what is a knockout ready or floxed allele?
tm1c
what is a lacZ reporter or constitutive knockout?
tm1b
what is a conditional knockout?
tm1d, which you get after breeding tm1c to a tissue-specific or inducible Cre
what would the cross be to get a homozygous mouse that expresses the reporter allele that is also null for the gene?
P0: GOI^tm1a/WT; WT x GOI^WT/WT; Cre
F1: GOI^tm1b/WT; Cre/WT GOI^WT/WT;Cre/WT GOI^WT/WT;Cre/WT GOI^WT/tm1b;Cre/WT
next you want to remove the Cre so you breed to a homozygous WT mouse
P0: GOI^WT/tm1b;Cre/WT x GOI^WT/WT;WT
F1: GOI^WT/WT;Cre/WT GOI^WT/WT;Cre/WT GOI^WT/tm1b;WT/WT GOI^WT/tm1b;WT/WT
this is the progeny chosen for sibling cross to get homozygous mutant
P0: GOI^WT/tm1b;WT/WT x GOI^WT/tm1b;WT/WT
F1: GOI^WT/WT;WT/WT GOI^WT/tm1b;WT/WT GOI^WT/tm1b;WT/WT GOI^tm1b/tm1b;WT
the last one is the homozygous mutant that has lacZ reporter and is null for the gene
how do you assign the sexes for the breeding strategy?
-you want the male to be the one that you put the mutants in
-they have the SRY factor that makes the progeny be males and can help get the changes into the germline
design a mouse that expresses the fuzzy-fluffy-eGFP fusion gene in the skin at 9 weeks of age in only male mice
-use XY allele that has a constitutive promoter upstream of a stop codon in between two loxP sites upstream of the GOI
-use a skin-specific Cre is bound to the ERT gene so that you can give tamoxifen at 9 weeks and then Cre will excise the stop codon and you get constitutive expression of the fuzzy-fluffy-eGFP fusion gene at 9 weeks
what are ways to temporally express Cre?
- CreERT2-Tamoxifen regulated
- Tetracycline Responsive Cre
Tamoxifen-inducible: Cre-ER^T2
Cre is under the promoter of estrogenic receptor –> whenever Tamoxifen is introduced, Cre is expressed and can go and act on site of interest
Tetracycline inducible system
-Tetracycline is administered and you have Cre under expression of Tet responsive element, which leads to expression of Cre
-not a great system since you have a lot of leakage
What are other ways to temporally regulate Cre expression?
virus and BAC
how do you screen for your mutations?
- floxed mice
- point mutant strategy
floxed allele
-use different primers for 5’ and 3’ loxP sites and exterior arrows will show the deletion
-put the arrows just before the loxP site and slightly above the exon that is being excised
point mutant strategy
-if you’re lucky enough to get a sequence that has a restriction site, you can use restriction fragment length polymorphism PCR
-cut genetic material with restriction enzyme and run it on a gel
