functional genomics Flashcards
what is functional genomics
The exploration of gene function on a global scale, focusing on the dynamic aspects such as gene transcription, translation, and protein-protein interactions
what is forward genetics
generate random mutations and look for phenotypes of interest
ideally do saturation mutagenesis, every possible locus is hit.
mutations must be analysed for all possible functions - behaviour, development etc.
reverse genetics
select gene of interest first, then knock it out
fine structure genetics
reverse but more specific
select gene of interest, knock-out or knock-in function in specific tissue and/or time point
what methods can be used to generate mutations at a large scale?
gamma rays - induce chr inversions, translocations, deletions
chemicals - point mutations, small deletions, non biased, detection is difficult.
insertions - DNA insertion (tagged) into the genome, non random distribution.
define: antimorph hypermorph neomorph hypomorph
antimorph - dom neg function
hypermorph - gain of function
neomorph - new function
hypomorph - haploinsufficient loss of function (mild phenotype, not full knock out)
what does it mean if a gene has embryonic lethal function?
genes needed for embryonic dev, also have another function in adult. IF KO, embryo dies so don’t see function in adult.
5 limitations of forward genetics
- Cannot identify maternal factors
- Cannot identify genes with redundantly specified function
- Cannot identify functions that are masked by embryonic lethal functions
- Mutations generated need to be mapped (deficiency mapping)
- Mutations generated need to be preserved (balancer chromosomes Drosophila)
what is a deficiency line
line with part of a chromosome missing
the missing part is known
what is deficiency mapping
cross homozygous mutant with many different deficiency lines, to create a lot of combinations.
if mutation loci is present in WT then no effect of mutation. however, if mutation loci is where the def line is missing, then the mutation is apparent in phenotype.
old school method
how does a balancer chromosome work?
prevents mutation getting lost over generations.
must be preserved in a heterozygous background, but crossing over can replace it with a WT allele.
balancer chr has one arm inverted to prevent crossing over of homologous car in meiosis. all genes are still present and functional. contains a gene which is lethal if homozygous for balancer chr.if mutation is lethal, then mutants which are homo for mutation also die, so left with progeny heterozygous and genotypes are known.
how can DNA insertion be used for large scale mutagenesis screens?
transposon insertion
randomly jump to a new location, but can choose where to initially insert it because they have specific flanking regions.
Inject p-element and helper element (encodes transposes) into blastoderm stage before cellularisation, and then these get incorporated into the germ line cells, so progeny with have
many mutations created per generation
site of mutation can be easily recovered - plasmid rescue (meaning no deficiency mapping needed to identify the locus)
how can we analyse and identify mutations in reverse genetics?
systematic mutagenesis - knock out a series of genes
Knock-ins - introduce modified version of target gene
RNAi - specific knock out of gene of interest
Gain of function mutagenesis - change gene product so it gains a new function
how can a specific gene be targeted?
transform organisms with specific constructs which can disrupt or replace the gene.
inhibit translation of target gene use other molecules to mimic or supress function
what is cre-lox recombination
site specific recombinase technology
specific cell type or triggered by stimulus
cre recombinase enzyme combines a pair or short sequences (lox seqs).
loxP is the original enzyme and sequence system, derived from bacteriophage P1.
can place lox sequences to manipulate genes.
4 methods of introducing new DNA into a cell
microinjection
electroporation/chemically
bacteria/virus
biolistics
3 methods of getting new DNA to stably integrate into the genome
illegitimate recombination
homologous recombination
transposable element
how can we transform plants using a natural system?
A. tumefaciens bacteria - has T1 plasmid with transformed T-DNA from previous action.
transfers T DNA into the plant genome.
Grow transformed cells on plate and culture transgenic plant.
what is RNAi briefly?
allows knock down of target genes using dsRNA copies of the gene.
likely an ancient defence system against RNA viruses.
describe 2 mechanisms of RNAi
dicer protein trims dsRNA to form siRNA or miRNA.
RISC - RNA-induced silencing complex. complex contains argonaute and other proteins. post transcriptional - targets mRNA
RITS - RNA induced transcriptional silencing. complex contains argonaute, chromodomain protein and Tas3. causes histone modification and heterochromatin formation.
if a gene has several functions which mask each other, how can you study it?
Gain of function mutagenesis
- express the gene under an inducible promoter (choose time point and tissue)
- express dominant loss of function form
- express constitutive active forms
what is ectopic expression
expression of a gene in a cell where it isn’t usually expressed
when is fine structure genetics useful?
functions are either masked by redundant loci and/or when certain regions are investigated.
Allows more in depth analysis of gene function.
what are 2 tools used for conditional mutagenesis (part of fine structure genetics)?
enhancer trapping
floxing
Allows KO or GoF at a desired time point/ tissue
Large scale screens w mass mating and driver/reporter lines
how does enhancer trapping work?
enhancer trap construct requires TE (for random insertion in genome) and reporter gene (id of spatial regulation). can also include a genetic marker (eg red eyes).
Allows identification of gene enhancers.
put the construct behind a gene with conditional expression and unknown enhancer. note which cells and who they are expressing the gene to characterise the enhancer.
However - reporter protein can linger after the enhancer is no longer active
what is the GAL4-UAS system?
many GAL4 lines exist - drosophila which express gal4 in different cell types. gal4 alone has very little effect. driver line includes enhancer and gal4 gene.
Reporter lines - strains of flies with UAS region next to a desired gene.
cross driver and reporter
gal4 expression = gal4 protein
gal4 activates UAS on reporter line, which controls expression of target gene
what is floxing?
cross line with loxP sites with line with cre recombinase, under a tissue specific enhancer
allows excision of genes in specific tissue types.
how can crispr be used for epigenetic regulation?
add molecule to Cas9 which modulates the epigenetic marks.
sgRNA specific to certain part of genomes can target certain genes epigenetic marks.
