large scale genetic screens in zebrafish

gene reg sem 2

what organisms do we use to observe development and morphological analysis and WHY

• zebrafish

• frog

• chicken

large eggs, accessible embryos, short development time and easy to keep in lab

what organisms do we use to manipulate the embryo and why

• frog

• chicken

large accessible embryos, robust embryos that can tolerate manipulation or embryos that can be grown in a dish, in culture

what organisms do we use for developmental genetics and why

• nematode worm and fruitfly (not vertebrates)

• zebrafish

• mouse (most common)

key advantages of using zebrafish

• speed (48hr from fertilisation to organ dev)

• size (large enough to be screened in dissecting microscopes but small enough to be kept in large vols)

• transparent

• high no of offspring (600 per clutch)

• genetic model

what is a genetic screen

a lab procedure used to create and detect a mutant organism (detect a change in organism created by mutation)

types of genetic screens

genetic screens can be split into 2 general groups

1. forward genetics

2. reverse genetics

forward genetics

classical approach of identifying a gene by its mutant phenotype FIRST

mutagenesis

genetic info is changed reuslting in mutation (can be spontaneous or as a result of exposure to mutagens)

chemical mutagenesis

dna is damaged by physical, chemical and biological agents (called mutagens)

• most common is ENU

phenotypic diffs appear in the offspring

map the damaged gene to assign a function

process of chemical mutagenesis using ENU (zebrafish)

put ENU in water with the fish in for 6 weeks

• the ENU causes mutations in the stem cells which generate new sperm (spermatogonia)

• creating a set of zebrafish where SOME are heterozygous for the mutation if they were fertilised by sperm which are produced from mutated spermatogonia

• then breed again to get fish homozygous for the mutation and then screen for phenotypic diff

largest scale mutagenesis screen ever done

• 1993

• identification of 1000 mutants affecting many processes

insertional mutagenesis

creation of genomic mutations thru addition of one or more base pairs

• integrate randomly into the genome

• map damaged gene and assign function

tol2 transposon

encodes a functional transposase which can catalyse the transposition of a transposon construct that has 200 and 150 bp of DNA from the left and right ends of ther Tol2 seq

• can do large inserts

• works in all vertbrates

enhancer trap (insertional mutagenesis)

truncates the protien and causes flourescence to see if insertion did anything

gene trap (insertional mutagenesis)

creates a fusion protien between the truncated protein and the inserted GFP

can see when and where a gene is expressed

insertional mutagenesis screens in zebrafish

• insertion of intervening region thru genome of fish

• injection of plasmid into zebrafish embryo along weith the tol2 mRNA

• breed with wild type

• causing a mixture of different expressions of GFP in diff areas

• then breed again to follow mutation an dget heterozygous

how to identify the trap cassette insertion site

1. isolate genomic DNA from fish carrying cassette and digest with a restriction endonuclease

2. circulaise genomic fragments by addition of ligase

3. perform PCR with primers that bind to seqs in the trap cassette

4. amplified seqs can be sequenced and a database containing the genomic DNA can be searched using this seq to find the location of the insertion

example of an insertional mutagenesis screen

• tcf7

• tTF important in fin dev

• fish homozygous for trap cassette insertion developed smaller fins

reverse genetics?

investigations on a known gene, esp by creation of loss of function mutants and or transgenesis to over express gene of interest

• known gene and try to create mutations or disrupt functions

transgenesis

intro of a novel gene into the genome

• micro injection of DNA plasmids into one-cell stage zebrafish embryos

• allows us to over express a protein of interest and monitor it slocalization and dynamics within cells

targeted mutagenesis

gene knock out or knock down technique

mirpholino oligonucleotides

gene knock down technique

spatial control of transgene expression

2 fish lines

• GAL4 driver line

• UAS reporter line

• breed to get expression of gene of interest

temporal control of transgene expression

heat chockl promotor attached to transgene to drive gene expression of transgene

targeted mutagenesis by CRISP/cas9

to target endogenous gene

• cause damage and repair to specific gene of interest site

• get gene to repair itself (NHEJ/HDR)

zebrafish embryos used to model human disease

• 70% of human genes have a zebrafish ortholouge

  • 84% of disease causing genes have a counter part in zebrafish

morpholino oligoniclerotides

technique before CAS9

• makes a complemnentary strand to the mRNA to stop gene expression

• helps examine what happens if you reduce proitein activity