Balancer chromosomes Flashcards
How do balancer chromosomes stop recombination?
They have multiple inversions which would make the homologous chromosomes form inversion loops in order to synapse and cross over, which is very unfavourable
What happens if a homologous chromosome does recombine with the homologous chromosome?
Creates inviable recombination products with deletions and duplications
How do we recognize that an organism has a balancer chromosome?
The balancer will carry a dominant mutant allele that can be selected for
How do we prevent homozygosity for the balancer chromosome?
It carries a recessively lethal allele that allows the balancer to exist in a heterozygous state, but not in a homozygous state. The dominant selection allele can also be the recessively lethal allele
Why don’t balancers for the X chromosome carry a recessively lethal allele?
Would prevent us from having a balancer stock of males, which we sometimes need
Which chromosome is FM7a a balancer for in Drosophila? What’s on it?
X chromosome. FM indicates “first multiple”. Carries the bar eyes mutation, which is dominant but not recessively lethal
Which chromosome is SM5 a balancer for in Drosophila? What’s on it?
Chromosome 2. SM indicates “second multiple”. Carries the curly wings mutation, which is dominant and recessively lethal
Which chromosome is TM6b a balancer for in Drosophila? What’s on it?
Chromosome 3. TM indicates “third multiple”. Carries the stubble bristles, which is dominant and recessively lethal
Why don’t we have any balancers for chromosome 4 in Drosophila?
Too small and not a lot of genetic material
What is a balancer stock?
A stock of female organisms that have one of the two copies of the chromosome of interest being the balancer chromosome, and the homolog has a dominant selectable marker
Why do we need to use virgin females for Drosophila?
They can store sperm, which would mess up the crosses, so they need to have never seen a male fly
Why does the homologous chromosome in the balancer stock need to have some sort of selectable marker?
We need to select against it later to make sure we have a mutagenized chromosome over the balancer later down the line in a mutant hunt
What is the first cross you would do in a mutant hunt with balancer chromosomes?
Cross the mutagenized males with balancer females
In a mutant hunt with balancer chromosomes, what do we select for and against in the F1 generation?
Select males with the balancer phenotype
In a mutant hunt with balancer chromosomes, what do we cross the selected F1 males with?
Individual crosses with the balancer stock females
In a mutant hunt with balancer chromosomes, what do we select for and against in the F2 generation? Why?
Select for the balancer phenotype and against the dominant mutation on the non-mutagenized homolog. If we select against the dominant mutation, we know that the balancer chromosome is coming from the female so the other chromosome could potentially contain a mutation
In a mutant hunt with balancer chromosomes, what do we cross the selected F2 males and females with?
Each other. If there is a recessive mutation, we could get it in a homozygous state
Are mutant hunts part of forward genetics or reverse genetics?
Forward genetics
In a mutant hunt with balancer chromosomes, what could it mean if we don’t see any individuals with the non-balancer phenotype in the F3 generation?
The mutation generated could be recessively lethal
How could we isolate other mutant alleles for the same gene?
Use a second balancer that is for the same chromosome, but can be distinguished from the other balancer. Cross the mutagenized males with balancer females (with the second balancer), then cross the balancer phenotype F1 with the balanced stock of the original mutant allele. If we get any individuals without the balancer phenotype, we have a new allele
Why is it trickier to use balancer chromosomes in mice vs in flies?
A lot more chromosomes
What two technologies have allowed us to create balancer chromosomes in mice?
Embryonic stem cell cultures and specific recombination systems (Cre/loxP)
Where does the Cre/loxP system come from?
P1 bacteriophage
What does Cre recombinase do?
Catalyzes recombination between 2 loxP sites
What is a loxP site?
A particular DNA sequence that has an asymmetrical 8 bp core region, flanked by 2 inverted 13 bp sequences
What will happen if Cre recombinase catalyzes recombination between two loxP sites that are in cis and oriented in the same direction?
Generates a deletion
What will happen if Cre recombinase catalyzes recombination between two loxP sites that are in cis and oriented in the opposite directions?
Generates an inversion
What will happen if Cre recombinase catalyzes recombination between two loxP sites that are in trans?
Generates a reciprocal translocation
What is special about the mouse embryonic stem cells that we culture to make balancer chromosomes?
They are homozygous mutant for the hprt gene and can’t metabolize hypoxanthine, so they can’t grow in HAT media
What are the components of the targeting vector to the breakpoint of the inversion of our new balancer chromosome?
Selectable marker: drug resistance, different one for both breakpoints
Either 5’ or 3’ end of the WT HPRT gene
LoxP site: in the intron of HPRT
Dominant selection allele: different one for both breakpoints
Terminal sequences that match the target loci so the vector gets incorporated in the right place
Why do we need to have different selectable markers for each breakpoint in the targeting vectors?
So we know the cell got both of the vectors and not just one
Why does each vector only have half of the WT HPRT gene?
If recombination occurs, the two halves of the gene get brought together and we get a functional gene product, so the cell with grow on HAT media
Why do the loxP sites need to be in the intron of the HPRT gene in the targeting vector?
To not disrupt the functional gene product
After creating the targeting vectors, what do we do with them?
Transfect them into embryonic stem cells that are hprt mutants
How do we select the embryonic stem cells that took up both vectors?
Grow them in the presence of both drugs that the vectors have resistance alleles for. Only the cells that took up both vectors will survive
What do we do with the cells that took up both vectors?
Transfect in another vector that expresses Cre recombinase
How do we know if recombination occurred in the cells with both vectors and expressing Cre recombinase?
Grow the cells on HAT media. If they had recombination, then they will have a functional HPRT gene due to the 3’ and 5’ ends being brought together
How do we get the cells with the balancer chromosome out of the cell culture and into a mouse?
Inject the transformed cells into another blastocyst before the cells get specified, then implant those into an albino female mouse and let them grow
What will the phenotype of the baby mice that formed from the injected blastocysts be?
Chimeric. They will have mottled coats, patches of normal colour with patches of agouti from the transformed cells
What do we do with the chimeric mice?
Mate them individually to normal coloured mice and see if we get a solid agouti coat
What does it mean if none of the progeny of a chimeric mouse have solid agouti coats?
The transformed cells did not end up in the germline cells
In a mutant hunt with mice, what is the first cross?
Cross mutagenized males with balancer females that have a dominant selection allele on the homolog
In a mutant hunt with mice, what phenotype do we select in the F1 mice?
Males with the balancer phenotype and select against the dominant selection allele on the homolog
In a mutant hunt with mice, what do we cross the selected F1 males with? What do we select for and against in the progeny?
Balancer stock females. Select males and females that have the balancer phenotype and against the dominant allele on the homolog
In a mutant hunt with mice, what do we cross the selected F2 mice with?
Each other. Could get the mutation in a homozygous state
In a mutant hunt with mice, what could be the explanation if we see no mice without the balancer phenotype in the F3 generation?
The mutations could be recessively lethal
