Drosophila vectors and transformation

Question 1

What are most Drosophila vectors based off of?

Answer 1

P-elements

Question 2

What mechanisms do P-elements use to move around in the genome?

Answer 2

Cut and paste/non-replicative transposition

Question 3

What does an endogenous P-element look like?

Answer 3

31 bp inverted repeats flanking 4 exons of transposase, exons labelled 0 to 3

Question 4

Which intron in an endogenous P-element with transposase shows alternative splicing? How does that affect the version of transposase in a tissue?

Answer 4

The intron between exons 2 and 3. Different splicing patterns in germline and somatic cells

Question 5

What splicing pattern of an endogenous P-element is seen in germline cells?

Answer 5

The entire intron is cut out and produces functional transposase, and the P-element moves around

Question 6

What splicing pattern of an endogenous P-element is seen in somatic cells?

Answer 6

The intron does not get cut out and contains an inframe stop codon, which encodes a repressor instead of transposase, which stops the P-element from moving around

Question 7

What is Delta2-3?

Answer 7

A constitutive source of transposase with the intron cut out, so we can express functional transposase in any tissue we want

Question 8

What is the difference between an M-cytotype and a P-cytotype fly?

Answer 8

M-cytotypes don’t have endogenous P-elements, so can’t encode the repressor. P-cytotypes do have the endogenous P-elements and can encode the repressor

Question 9

What happens if we cross an M-cytotype female to a P-cytotype male?

Answer 9

Produce dysgenic progeny that is often sterile with high rates of mutation

Question 10

Why do we get dysgenic progeny when crossing an M-cytotype female to a P-cytotype male?

Answer 10

The egg doesn’t have any P-elements to encode the repressor, so the P-elements move around completely uncontrolled and create tons of mutations

Question 11

What happens if we cross a P-cytotype female to an M-cytotype male?

Answer 11

Get normal progeny since the repressors from the female control the P-elements

Question 12

What are autonomous P-elements?

Answer 12

P-elements that can move themselves and move other stuff in trans

Question 13

What are non-autonomous P-elements?

Answer 13

P-elements where most of transposase is deleted, but the inverted repeats are intact

Question 14

What is on a fly vector, like the pCaSpeR plasmid?

Answer 14

Bacterial ori, bacterial selectable marker on the backbone. Has the inverted repeats, and a WT sequence for the white gene (selectable marker) and the MCS between them

Question 15

What are 3 motivations for transforming flies?

Answer 15

Generate mutants, determine the phenotypic consequences of a sequence, generate deletions by improper excision

Question 16

What are 3 possible disruptions that be generated by P-element insertions?

Answer 16

Insertion into exon: likely will generate non-functional gene product
Insertion into intron: may interfere with splicing or do nothing
Insertion into regulatory sequences: may impact expression

Question 17

What are the 3 things we need to have for fly transformation?

Answer 17

1. A functional source of transposase
2. a pair of intact inverted repeats enclosing a selectable marker
3. A way to prevent continuous P-element movement once it has jumped into the genome

Question 18

What is a wings clipped plasmid?

Answer 18

Contains a functional transposase gene but no inverted repeats flanking it, so transposase can move other stuff in trans but not move itself

Question 19

What are two possible ways to start out fly transformation?

Answer 19

Either inject an embryo with an endogenous transposase, or do binary transformation

Question 20

If we are planning on injecting embryos with an endogenous source of transposase, how do we create that embryo?

Answer 20

Start with a male that has white eyes and Delta2-3 and a dominant mutation (Ki) on one homolog (can be on any chromosome), and cross it to a white-eyed female with two different balancers for chromosome 3 (or whatever other chromosome the transposase is on). Then you inject those embryos with a plasmid containing w+ and YFG between the inverted repeats

Question 21

After injecting embryos with an endogenous source of transposase, which of the adults do we select?

Answer 21

Select for kinked bristles, since those ones will have transposase that could also be in the germline cells

Question 22

What do we cross the selected G0 adults with kinked bristles to?

Answer 22

White eyed flies, male or female

Question 23

What do we select from the G1 adults after crossing kinked bristles with white eyed flies? Why?

Answer 23

Select for red eyes and the balancer phenotype and no kinked bristles. These flies had the P-element move in and now we want to remove the transposase so it doesn’t keep moving

Question 24

What is binary transformation?

Answer 24

Injecting 2 plasmids into an embryo without an endogenous source of transposase

Question 25

What is on the two plasmids for binary transformation?

Answer 25

One plasmid has selectable marker (w+) and YFG between the inverted repeats, and the other has a wings clipped transposase

Question 26

What embryos are we injecting for binary transformation?

Answer 26

Homozygous w- embryos that are the offspring between 2 true breeding white eyed parents

Question 27

What do we cross the injected G0 embryos to in binary transformation (once they become adults)?

Answer 27

True breeding white eyed flies

Question 28

What do we select from the G1 generation in binary transformation?

Answer 28

Red eyes - shows they got the P-element

Question 29

What is P-element mobilization?

Answer 29

The stock flies already have a P-element somewhere in their genome already, and we want to move it

Question 30

What is the first cross to do when doing P-element mobilization?

Answer 30

Cross a red eyed male with a dominant selection allele on the same chromosome as the P-element (L), that got the red eyed phenotype from a P-element to a white eyed female with transposase and a dominant selection marker (delta2-3, Ki) and a balancer across from that

Question 31

What progeny do we select in the F1 when doing P-element mobilization?

Answer 31

Males with red lobed eyes and kinked bristles

Question 32

What do we cross the selected F1 males to in P-element mobilization?

Answer 32

True breeding white eyed females

Question 33

What do we select from the F2 generation in P-element mobilization?

Answer 33

Males with red eyes, and select against lobe eyes and stubble bristles

Question 34

What is the first step to determine which chromosome a P-element moved to after P-element mobilization?

Answer 34

Start by crossing the males with the mobilized P-element to white-eyed females with a balancer and a homolog with a dominant mutation for chromosomes 2 and 3

Question 35

How do we know if the P-element moved to the X chromosome?

Answer 35

We can tell from the first cross. All the males will have white eyes and all the females will have red eyes

Question 36

How do we know if the P-element moved to chromosome 2?

Answer 36

We need to do a second cross. Cross selected males with red eyes and both balancer phenotypes with the injection strain (white eyed females with no balancers). If the P-element was on chromosome 2, there will be no flies with both red eyes and the chromosome 2 balancer phenotype

Question 37

How do we know if the P-element moved to chromosome 3?

Answer 37

We need to do a second cross. Cross selected males with red eyes and both balancer phenotypes with the injection strain (white eyed females with no balancers). If the P-element was on chromosome 3, there will be no flies with both red eyes and the chromosome 3 balancer phenotype