Genet 302 lec 25 & 26
What is the goal of a conditional mutation?
Only the target tissue (dispensable tissue) is mutant while the rest of the mouse stays viable and fertile
What is a site-specific recombinase system?
A system where an enzyme causes recombination at specific DNA target sites in somatic cells- not during mitosis
What are the target sites and enzyme in the mouse conditional system?
LoxP sites and Cre recombinase
Where do the Cre/Lox components come from?
Lox and Cre come from P1 phage
What does Cre do when 2 loxP sites flank a DNA segment in the same orientation?
It recombines the loxP sites and deletes the DNA between them
What does floxed mean?
Flanked by loxP sites
What is a floxed mouse?
A mouse in which important exon(s) of a gene are flanked by loxP sites
Why are important exon(s) chosen for floxing?
Because deleting them will disrupt the gene and create a knockout in cells where Cre is expressed
How is a floxed strain of YFG made?
Use CRISPR + HDR to insert one loxP site on each side of important exon(s) of YFG
How many guide RNAs and donor DNAs are needed to make a floxed allele in this lecture?
Two guide RNAs and two donor DNAs
Why are 2 guide RNAs needed to make a floxed allele?
One targets one side of the exon and the other targets the other side
Why are 2 donor DNAs needed to make a floxed allele?
One donor inserts the first loxP site and the other donor inserts the second loxP site
How would you diagram making a floxed strain of YFG?
Show YFG with important exon(s), Cas9 plus 2 gRNAs cutting on each side, 2 donor DNAs carrying loxP sites, HDR inserting both loxP sites, and the final floxed YFG allele
How are Cre mice made in lecture 25?
By pronuclear injection
What is the basic DNA construct used to make a Cre mouse?
A tissue-specific promoter driving Cre cDNA
Why is the promoter important in a Cre mouse?
It determines where Cre will be expressed
How are floxed mice used to make a conditional mutation?
Cross the floxed YFG mouse with a transgenic Cre mouse that expresses Cre in a specific tissue
What happens in offspring that inherit both the floxed YFG allele and the Cre transgene?
Cre deletes the floxed exon(s) only in the cells where the Cre promoter is active
How would you diagram using a floxed YFG strain to make a conditional mutation?
Show a floxed YFG mouse crossed with a tissue-specific Cre mouse, then in double-transgenic offspring show Cre expressed only in the target tissue and deletion of the floxed exon only there
What is the difference between a floxed allele and a conditional knockout?
A floxed allele is still functional until Cre is expressed, while a conditional knockout is produced after Cre deletes the floxed exon(s) in specific cells
What syndrome is the lecture 26 mouse model related to?
Phelan-McDermid syndrome
What kind of disorder is Phelan-McDermid syndrome in this lecture?
A contiguous gene disorder
What chromosome region is deleted in Phelan-McDermid syndrome?
22q13.3 : can be terminal or interstital
What gene is emphasized as causing the autosomal dominant CNS problems in this syndrome?
SHANK3
What do SHANK3 proteins do?
They connect receptors to the cytoskeleton at synapses
What mouse strain was first made in the Shank3 example?
A Shank3 floxed strain
What was flanked by loxP sites in the Shank3 floxed strain?
Most of the Shank3 gene, effectively exons 4 through 22
What was the second strain used to convert the floxed Shank3 allele into a knockout?
A CMV-Cre strain
What type of promoter drives Cre in the CMV-Cre strain?
A constitutive promoter active in most cells and tissues
Why does CMV-Cre make a full knockout rather than a tissue-specific conditional mutation?
Because Cre is expressed in most cells, including germ cells
How was the Shank3Δ4-22 knockout strain generated from the floxed strain?
By crossing the Shank3 floxed mouse with the CMV-Cre mouse so Cre deleted the loxP-flanked region
How would you use a floxed strain of YFG to make a knockout mutation?
Cross the floxed YFG strain to a constitutive Cre strain, let Cre delete the loxP-flanked exon(s) in all cells, then breed and identify mice carrying the deleted YFG allele
What is PCR used for in verifying a mouse strain?
To amplify specific DNA regions that differ between wild-type, floxed, and knockout alleles
What does AGE stand for?
Agarose gel electrophoresis
What does agarose gel electrophoresis do in this context?
Separates PCR products by size so different alleles can be distinguished as different bands
How do primer positions help verify a mouse strain?
Different primer pairs amplify different allele structures, so band presence and size reveal the genotype
Why can a heterozygous mouse give multiple PCR bands?
Because it carries 2 different alleles that generate different PCR products
How many bands were expected in heterozygous mice in the Shank3Δ4-22 verification slide?
Two?
How would you diagram PCR + AGE to verify a mouse strain?
Show genomic DNA, primers flanking diagnostic regions, PCR amplification of allele-specific fragments, loading products on an agarose gel, and different band patterns for WT, heterozygote, and knockout
What 3 lecture methods are linked together across lectures 25 and 26 to make and verify these strains?
CRISPR to make the floxed allele, Cre/Lox to delete it, and PCR plus agarose gel electrophoresis to verify the genotype
What phenotype pattern was shown for the Shank3Δ4-22 mice in the buried food test?
KO mice were much slower than WT and heterozygotes, while heterozygotes were closer to WT
What phenotype pattern was shown for the Shank3Δ4-22 mice in the marble burying test?
KO mice buried far fewer marbles than WT and heterozygotes, while heterozygotes were close to WT
What inheritance pattern was suggested by the behavioral results shown for the Shank3Δ4-22 mouse strain?
Autosomal recessive-like for the measured mouse behavioral phenotypes