Balancer chromosomes

Question 1

How do balancer chromosomes stop recombination?

Answer 1

They have multiple inversions which would make the homologous chromosomes form inversion loops in order to synapse and cross over, which is very unfavourable

Question 2

What happens if a homologous chromosome does recombine with the homologous chromosome?

Answer 2

Creates inviable recombination products with deletions and duplications

Question 3

How do we recognize that an organism has a balancer chromosome?

Answer 3

The balancer will carry a dominant mutant allele that can be selected for

Question 4

How do we prevent homozygosity for the balancer chromosome?

Answer 4

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

Question 5

Why don’t balancers for the X chromosome carry a recessively lethal allele?

Answer 5

Would prevent us from having a balancer stock of males, which we sometimes need

Question 6

Which chromosome is FM7a a balancer for in Drosophila? What’s on it?

Answer 6

X chromosome. FM indicates “first multiple”. Carries the bar eyes mutation, which is dominant but not recessively lethal

Question 7

Which chromosome is SM5 a balancer for in Drosophila? What’s on it?

Answer 7

Chromosome 2. SM indicates “second multiple”. Carries the curly wings mutation, which is dominant and recessively lethal

Question 8

Which chromosome is TM6b a balancer for in Drosophila? What’s on it?

Answer 8

Chromosome 3. TM indicates “third multiple”. Carries the stubble bristles, which is dominant and recessively lethal

Question 9

Why don’t we have any balancers for chromosome 4 in Drosophila?

Answer 9

Too small and not a lot of genetic material

Question 10

What is a balancer stock?

Answer 10

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

Question 11

Why do we need to use virgin females for Drosophila?

Answer 11

They can store sperm, which would mess up the crosses, so they need to have never seen a male fly

Question 12

Why does the homologous chromosome in the balancer stock need to have some sort of selectable marker?

Answer 12

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

Question 13

What is the first cross you would do in a mutant hunt with balancer chromosomes?

Answer 13

Cross the mutagenized males with balancer females

Question 14

In a mutant hunt with balancer chromosomes, what do we select for and against in the F1 generation?

Answer 14

Select males with the balancer phenotype

Question 15

In a mutant hunt with balancer chromosomes, what do we cross the selected F1 males with?

Answer 15

Individual crosses with the balancer stock females

Question 16

In a mutant hunt with balancer chromosomes, what do we select for and against in the F2 generation? Why?

Answer 16

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

Question 17

In a mutant hunt with balancer chromosomes, what do we cross the selected F2 males and females with?

Answer 17

Each other. If there is a recessive mutation, we could get it in a homozygous state

Question 18

Are mutant hunts part of forward genetics or reverse genetics?

Answer 18

Forward genetics

Question 19

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?

Answer 19

The mutation generated could be recessively lethal

Question 20

How could we isolate other mutant alleles for the same gene?

Answer 20

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

Question 21

Why is it trickier to use balancer chromosomes in mice vs in flies?

Answer 21

A lot more chromosomes

Question 22

What two technologies have allowed us to create balancer chromosomes in mice?

Answer 22

Embryonic stem cell cultures and specific recombination systems (Cre/loxP)

Question 23

Where does the Cre/loxP system come from?

Answer 23

P1 bacteriophage

Question 24

What does Cre recombinase do?

Answer 24

Catalyzes recombination between 2 loxP sites

Question 25

What is a loxP site?

Answer 25

A particular DNA sequence that has an asymmetrical 8 bp core region, flanked by 2 inverted 13 bp sequences

Question 26

What will happen if Cre recombinase catalyzes recombination between two loxP sites that are in cis and oriented in the same direction?

Answer 26

Generates a deletion

Question 27

What will happen if Cre recombinase catalyzes recombination between two loxP sites that are in cis and oriented in the opposite directions?

Answer 27

Generates an inversion

Question 28

What will happen if Cre recombinase catalyzes recombination between two loxP sites that are in trans?

Answer 28

Generates a reciprocal translocation

Question 29

What is special about the mouse embryonic stem cells that we culture to make balancer chromosomes?

Answer 29

They are homozygous mutant for the hprt gene and can’t metabolize hypoxanthine, so they can’t grow in HAT media

Question 30

What are the components of the targeting vector to the breakpoint of the inversion of our new balancer chromosome?

Answer 30

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

Question 31

Why do we need to have different selectable markers for each breakpoint in the targeting vectors?

Answer 31

So we know the cell got both of the vectors and not just one

Question 32

Why does each vector only have half of the WT HPRT gene?

Answer 32

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

Question 33

Why do the loxP sites need to be in the intron of the HPRT gene in the targeting vector?

Answer 33

To not disrupt the functional gene product

Question 34

After creating the targeting vectors, what do we do with them?

Answer 34

Transfect them into embryonic stem cells that are hprt mutants

Question 35

How do we select the embryonic stem cells that took up both vectors?

Answer 35

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

Question 36

What do we do with the cells that took up both vectors?

Answer 36

Transfect in another vector that expresses Cre recombinase

Question 37

How do we know if recombination occurred in the cells with both vectors and expressing Cre recombinase?

Answer 37

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

Question 38

How do we get the cells with the balancer chromosome out of the cell culture and into a mouse?

Answer 38

Inject the transformed cells into another blastocyst before the cells get specified, then implant those into an albino female mouse and let them grow

Question 39

What will the phenotype of the baby mice that formed from the injected blastocysts be?

Answer 39

Chimeric. They will have mottled coats, patches of normal colour with patches of agouti from the transformed cells

Question 40

What do we do with the chimeric mice?

Answer 40

Mate them individually to normal coloured mice and see if we get a solid agouti coat

Question 41

What does it mean if none of the progeny of a chimeric mouse have solid agouti coats?

Answer 41

The transformed cells did not end up in the germline cells

Question 42

In a mutant hunt with mice, what is the first cross?

Answer 42

Cross mutagenized males with balancer females that have a dominant selection allele on the homolog

Question 43

In a mutant hunt with mice, what phenotype do we select in the F1 mice?

Answer 43

Males with the balancer phenotype and select against the dominant selection allele on the homolog

Question 44

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?

Answer 44

Balancer stock females. Select males and females that have the balancer phenotype and against the dominant allele on the homolog

Question 45

In a mutant hunt with mice, what do we cross the selected F2 mice with?

Answer 45

Each other. Could get the mutation in a homozygous state

Question 46

In a mutant hunt with mice, what could be the explanation if we see no mice without the balancer phenotype in the F3 generation?

Answer 46

The mutations could be recessively lethal