Drosophila vectors and transformation Flashcards
What are most Drosophila vectors based off of?
P-elements
What mechanisms do P-elements use to move around in the genome?
Cut and paste/non-replicative transposition
What does an endogenous P-element look like?
31 bp inverted repeats flanking 4 exons of transposase, exons labelled 0 to 3
Which intron in an endogenous P-element with transposase shows alternative splicing? How does that affect the version of transposase in a tissue?
The intron between exons 2 and 3. Different splicing patterns in germline and somatic cells
What splicing pattern of an endogenous P-element is seen in germline cells?
The entire intron is cut out and produces functional transposase, and the P-element moves around
What splicing pattern of an endogenous P-element is seen in somatic cells?
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
What is Delta2-3?
A constitutive source of transposase with the intron cut out, so we can express functional transposase in any tissue we want
What is the difference between an M-cytotype and a P-cytotype fly?
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
What happens if we cross an M-cytotype female to a P-cytotype male?
Produce dysgenic progeny that is often sterile with high rates of mutation
Why do we get dysgenic progeny when crossing an M-cytotype female to a P-cytotype male?
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
What happens if we cross a P-cytotype female to an M-cytotype male?
Get normal progeny since the repressors from the female control the P-elements
What are autonomous P-elements?
P-elements that can move themselves and move other stuff in trans
What are non-autonomous P-elements?
P-elements where most of transposase is deleted, but the inverted repeats are intact
What is on a fly vector, like the pCaSpeR plasmid?
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
What are 3 motivations for transforming flies?
Generate mutants, determine the phenotypic consequences of a sequence, generate deletions by improper excision
What are 3 possible disruptions that be generated by P-element insertions?
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
What are the 3 things we need to have for fly transformation?
- A functional source of transposase
- a pair of intact inverted repeats enclosing a selectable marker
- A way to prevent continuous P-element movement once it has jumped into the genome
What is a wings clipped plasmid?
Contains a functional transposase gene but no inverted repeats flanking it, so transposase can move other stuff in trans but not move itself
What are two possible ways to start out fly transformation?
Either inject an embryo with an endogenous transposase, or do binary transformation
If we are planning on injecting embryos with an endogenous source of transposase, how do we create that embryo?
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
After injecting embryos with an endogenous source of transposase, which of the adults do we select?
Select for kinked bristles, since those ones will have transposase that could also be in the germline cells
What do we cross the selected G0 adults with kinked bristles to?
White eyed flies, male or female
What do we select from the G1 adults after crossing kinked bristles with white eyed flies? Why?
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
What is binary transformation?
Injecting 2 plasmids into an embryo without an endogenous source of transposase
What is on the two plasmids for binary transformation?
One plasmid has selectable marker (w+) and YFG between the inverted repeats, and the other has a wings clipped transposase
What embryos are we injecting for binary transformation?
Homozygous w- embryos that are the offspring between 2 true breeding white eyed parents
What do we cross the injected G0 embryos to in binary transformation (once they become adults)?
True breeding white eyed flies
What do we select from the G1 generation in binary transformation?
Red eyes - shows they got the P-element
What is P-element mobilization?
The stock flies already have a P-element somewhere in their genome already, and we want to move it
What is the first cross to do when doing P-element mobilization?
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
What progeny do we select in the F1 when doing P-element mobilization?
Males with red lobed eyes and kinked bristles
What do we cross the selected F1 males to in P-element mobilization?
True breeding white eyed females
What do we select from the F2 generation in P-element mobilization?
Males with red eyes, and select against lobe eyes and stubble bristles
What is the first step to determine which chromosome a P-element moved to after P-element mobilization?
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
How do we know if the P-element moved to the X chromosome?
We can tell from the first cross. All the males will have white eyes and all the females will have red eyes
How do we know if the P-element moved to chromosome 2?
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
How do we know if the P-element moved to chromosome 3?
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
