C. Elegans Flashcards
what are the two sexes of C. elegans?
- hermaphrodites (XX)
- males (X0)
-hermphrodites crossed with males will give ~50% males
-hermaphrodites can also self fertilize and produce a large number of hermaphrodites
designing a screen to ID the anchor cell signal
-use WT worms, regular plates with OP50 bacteria for C. elegans to eat, and EMS (mutations all over the genome)
-look for vulvaless phenotype
-look at F2 generation for mutants and F1 has the dominant mutations
-this is considered a screen since you have to look at every worm
screen for bag of worms
P0: give the homozygous WT C. elegans EMS and allow them to self
F1: m/+ and allow them to self
F2: +/+ m/+ m/m and look for bag of worms among F2 progeny (the homozygous mutant)
isolating suppressors of a Muv mutation
EMS
Muv/Muv; +/+
Muv/Muv; m/+
Muv/Muv;+/+ Muv/Muv;m/+ Muv/Muv;m/m
-look for eggs on plates (know that it came from normal worms and it turns a screen to a selection)
-Non-Muv revertants in F1 or F2
-considered a selection since it’s high throughput
-faster way to find suppressor of mutation
epistasis
-genetic test where you take mutations with opposite phenotypes and combine them to make a double mutant and ask what is a phenotype of the double mutant?
-epistatic gene tells you the order of the two genes in the pathway
epistatic analysis example
-normal phenotypes from double mutants are not informative
-if phenotype of the double mutant is the same as both nulls of the single mutants, also not helpful
-compare the phenotype of the double mutant to the phenotype of each of the single null mutants –> whichever one it is, that gene is downstream of the other gene
-opposite phenotypes in null mutants then it is inhibited between them vs the same phenotype means activated
-if the phenotypes you are scoring are terminal phenotypes then the epistatic gene is the downstream gene
-if one of the phenotypes is an “intermediate” phenotype, the epistatic gene is the upstream gene
forward genetics
-drawback- gene affected is not known
-how can you ID a gene when all have is a mutant?
-out-cross mutant against a polymorphic strain
-use whole genome sequencing to simultaneously map and ID the mutation
hawaiian outcross and whole genome sequencing
-you can’t just sequence the mutant –> mutation in gene of interest but also other mutations that were generated from EMS
-shuffle genome and try to select for mutant by crossing mutant to a strain of worms that has polymorphism throughout the genome
-cross the Bristol and Hawaiian –> get F1 and generate F2s –> select for mutant in F2 again –> generate recombinant F2s because of recombination in the mother’s germline, the blue and yellow chromosomes have recombined
-sequence all of these genomes and only place where you see the Bristol signature of polymorphism is where mutant lies
-you want to ID F2s that display mutant phenotype and only evaluate those worms otherwise you will have F2 worms that are heterozygous and homozygous for the Hawaiian strain
-everywhere else you will see an equal distribution of Bristol and Hawaiian polymorphisms
genome editing
-repair of CRISPR/Cas9 cuts by HDR
-re-create mutations in WT background using Cas9
-introduce Cas9, targeting it to a particular gene to create a double stranded break and then @ the same time providing an oligo to repair that cut
-put in oligo the EMS mutation you sequenced
-if the mutation is recessive, after you make the CRISPR mutation, you will need to let F1 animals self and evaluate F2s for the phenotype
RNA-mediated interference
-feed dsRNA siRNA against a particular gene in bacteria and it will knock down that gene
-screen the entire genome of C. elegans
-know which genes you’re targeting –> do this with whole genome or selected group of genes
-can’t discover new genes with this method since you would need to know the gene of interest
CRISPR vs RNAi
-RNAi isn’t as precise as CRISPR so this is a knockdown rather than a knockout
reverse genetics
-gene is already known
-preselected group of genes that can have homology and are expressed in a specific tissue
-can also target annotated genes
EMS, RNAi, or CRISPR
-you are looking for genes that confer sensitivity to arsenic –> RNAi screen
-you want to screen all kinases –> RNAi screen
-you want to create a C. elegans model of a human genetic disease –> CRISPR
-you want to screen the whole genome for genes required for RNAi –> EMS
reverse vs forward genetics
reverse
-screen whole genome systematically
-no need to clone
-only knock-down (no special mutations)
-screen, not selection
-only predicted genes
forward
-mutagen bias
-cloning needed
-dominant, ts, partial
-selections increase efficiency
-not restricted to predicted genes
