Drosophila

Question 1

why are flies advantageous to study?

Answer 1

-they have many genes that are homologous to humans
-produce a lot of progeny
-easy to maintain

Question 2

chromosomal structure

Answer 2

-they have four chromosomes but we only care about the first three since the fourth one is so small it doesn’t matter
-the first chromosome is a sex chromosome and the other two are autosomes
-large number of available mutants
-small genome

Question 3

nomenclature

Answer 3

-genes and genetic elements are italicized
-gene names are abbreviated
-recessive mutants are lowercase: white, w and hedgehog, h
-allele names are superscripted
-dominant names are Capitalized
-; separates chromosomes
-, separates alleles
-+ WT alleles
—- separates homologous chromosomes
-Y chromosome can be a horizontal line with a slash

Question 4

sexes for fly crosses

Answer 4

-want to breed mutant male with virgin female
-if you know the genotype of your males, you can start with multiple males
-if you do not know the genotype of the male, start with a single one so you know what mutation you are working with
-once you get mutations, single male
-after you isolate one mutation, you can use multiple males and females again to optimize the amount of flies you get
-usually they will tell you where certain mutations are located and that will help you with determining chromosome structure and the sex of the parent
-mutants in male also because they do not go through recombination

Question 5

classical forward genetic screen

Answer 5

-screen for genes on the second chromosome essential for photoreceptor function
-choose EMS to generate random point mutations (CRISPR is not currently efficient for screening in Drosophila) –> unbiased, you can reach saturation, and get a bunch of mutants but it is slow for gene ID and large scale
-mutagenize males since they produce many gametes and recombination only occurs in females

Question 6

what are the characteristics of a balancer chromosome?

Answer 6

1. dominant marker
2. maintain recessive lethal mutations
3. have multiple inversions that prevent recombination

Question 7

balancer nomenclature

Answer 7

F=First; S=Second;T=Third

Question 8

what do balancers do?

Answer 8

help keep heterozygous mutations in a population by preventing recombination and having a marker that will allow us to keep track of the mutations in the population

Question 9

crossing flies with balancers

Answer 9

-in reality, every single female fly will recombine chromosomes and you won’t maintain these two different mutations

Question 10

GAL4/UAS binary system

Answer 10

-enables tissue- and cell-specific manner
-GAL4 TF is under the control of an enhancer (this enables the tissue specificity) and when it’s produced it will bind to a UAS sequence and whatever is downstream of the UAS will be expressed

Question 11

GAL4/UAS in flies

Answer 11

-in flybase, you see there is a GAL4 enhancer trap in the 5’ region of eos and it’s called eos-GAL4
-you cross eos-GAL4 flies to flies carrying a UAS-GFP reporter
-how can you verify that this accurately reflects the expression of eos? you can look for GFP expression and verify with an anti-eos antibody, FISH, or other reporter

Question 12

flip out clones vs mitotic clones

Answer 12

-flip out clones you have the FRT sites in the same gene, so there is no need for an additional chromosome
-mitotic clones you rely on recombination and have an additional chromosome with the second FRT site in the same position as the first one with GFP or some other visible marker

Question 13

FLP/FRT system can be used to edit the genome of individual cells, clones

Answer 13

-recombinase has target that it hunts for in the genome and catalyzes recombination and ligation
-directionality to it –> two of the FRT sites must be lying in the same direction
-apply heat shock to induce expression of the recombinase
-in the presence of FLPase the gene inside of the FRT sites will be excised

Question 14

FLP/FRT induced mitotic recombination efficiently generates mosaic tissues

Answer 14

-FRT sites are encoded on homologous chromosomes at the same genetic locus
-start with heterozygous cells and end up with two cells that are not the same genotype as the cell they came from
-end up GFP-negative mutant clone and GFP-positive twin spot

Question 15

which mutant is/are not amenable to the FLP/FRT mitotic recombination?

Answer 15

those that are upstream of the FRT sites

Question 16

markings of clones

Answer 16

1. positive- expressed brightly since they are homozygous GFP+
2. negative- expressed darkly since they are homozygous GFP-

Question 17

cell autonomous vs cell non-autonomous

Answer 17

-autonomous if the phenotype only affects the cell that it’s within and has neighboring WT cells
-non-autonomous if the phenotype changes the neighboring cells

Question 18

how do clones allow for mosaic expression?

Answer 18

-you can have homozygous GFP- cells
-you can have homozygous GFP+ cells
-you can have heterozygous GFP+/GFP- cells
-all of these will give different colors and they can appear next to each other giving the look of a mosaic and you can use the colors to determine the genotype

Question 19

conditional loss of expression using FLPase causes mosaic loss of expression

Answer 19

FLPing out a transgene will give you a patch of clones in dividing cells and will work in clump of non-mitotic cells because you don’t need to segregate these chromosomes

Question 20

different applications of clonal analysis methods

Answer 20

-use flip clones for tissue-specific expression
-use flip clones to create positively marked clones
-use mitotic clones to create cells that are homozygous for a mutant allele of eos

Question 21

what two components do you need for CRISPR in flies?

Answer 21

1. gRNA plasmid with an ORI and U6 promoter
2. homology-directed repair template with the mutation of interest and an ORI with a marker of interest, so you can have a novel restriction site or GFP

Question 22

how do you verify that the germline was modified?

Answer 22

mate single male flies to virgin females and extract gDNA from each fly stock and perform restriction digest based on the restriction site you inserted –> you will see bands that show if the restriction site was successfully cut or not

Question 23

what is the major drawback of tissue-specific (GAL4-driven) CRISPR?

Answer 23

you can’t guarantee that you will make a homozygous edit in every cell –> this is why you have screen using the marker that you put on the gRNA plasmid