Large scale genetic screens in zebrafish II

Question 1

8How are fish mutated witn ENU?

Answer 1

Male fish are put into water that has ENU added

Question 2

How does ENU work?

Answer 2

Transfers ethyl groups to nucleobases (usually thymidine) in nucleic acids

Question 3

What are the targets of mutation?

Answer 3

Spermatogonial stem cells–> derive mature sperm

Question 4

How many spermatogonial germ cells does each male fish have?

Answer 4

100-200

Question 5

WHat generation is the mutated male?

Answer 5

F0

Question 6

What generation are the offspring of the mutated male?

Answer 6

F1

Question 7

Genotype of the F1 generation?

Answer 7

Heterozygous

Question 8

How is there diff mutations in most of the offspring?

Answer 8

each come from a diff sperm cell, and diff sperm cells had diff mutations

Question 9

How is the mutation usually inherited in the F1 genertaion?

Answer 9

Usually recessive, so the F1 generation wont have the phenotype–> will be carriers

Question 10

What is done with the F1 generation?

Answer 10

Crossed to another wild type female

Question 11

Result of crossing an f1 generation fish with a WT female?

Answer 11

Half the offspring have the mutation (still recessive tho?

Question 12

What is done with the F2 generation?

Answer 12

They are crossed randomly

Question 13

What is the result of randomly crossing the F2 generation?

Answer 13

Half of its offspring have the mutation

Question 14

Why is the F2 generation created?

Answer 14

To have a generation for each mutation that is heterozygous for that specific mutaiton
I.e. can have multiple fish with the same mutation, so can then cross those to get a homozygous fish showing the mutated phenotype

Question 15

What is done with the F3 generation?

Answer 15

Observed for displaying the mutated phenotype

Question 16

What proportion of the F3 generation have the mutation?

Answer 16

1/4

Question 17

What is insertional mutagenesis?

Answer 17

Creation of genomic mutations through addition of one or more base pairs

Question 18

How can insertional mutagenesis occur naturally?

Answer 18

Viruses, transposons

Question 19

2 forms of insertional mutagenesis?

Answer 19

Enhancer trap and gene trap

Question 20

What is the Tol2 transposon?

Answer 20

ENcodes a fully functional transposase that has been modified to generate a mutagenesis approach

Question 21

What does the transposase protein do?

Answer 21

Catalyses the transposition of a transposon construct that has 200 and 150 BP of DNA from the left and right ends of the Tol2 sequence

Question 22

Which organisms has the Tol2 transposon been shown to work in?

Answer 22

zebrafish, xenopus, chicken, mouse, and human

Question 23

First step of using the Tol2 transposon?

Answer 23

Transposase mRNA and plasmid DNA w/ transposon construct are coinjected into a fertilised egg.

Question 24

What happens after the transposase and the DNA are injected into a fertilised egg?

Answer 24

They recombine and inserts into the genome

Question 25

What is done after the transposon inserts into the genome?

Answer 25

The eggs are grown, and screened to ID if things have entered genes of interest

Question 26

Components of an enhancer trap?

Answer 26

Minimal promoter, GFP, polyA tail

Question 27

What happens if an enhancer trap is inserted into a gene?

Answer 27

The neighboring enhancer (of the original gene) interacts with the minimal promoter of the gene and drives GFP expression (and maybe a truncated version of the protein)

Question 28

What does a gene trap consist of?

Answer 28

Splice acceptor, GFP, polyA tail

Question 29

What happens if a gene trap is inserted downstream of an exon?

Answer 29

It "traps" the gene so the protein produced is a fusion protein--> w/ a GFP tag