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DISCOVERY OF A GENE FUNCTION - Coggle Diagram
DISCOVERY OF A GENE FUNCTION
Forward genetics
Reverse genetics
Knock-out
Bacteria
In vitro transposition
: inducing transposition with a DNA target, transposase and transposon.
GAMBIT
: genome is fragmented; these fragments transform into the bacteria in vivo; you grow the bacteria treated and then you make a PCR with a transposon specific primer and a chromosomal primer; you make PCR on the mix of bacterial DNA and then you get different amplified fragments whose length may vary
Yeast
S. cerevisiae
S. pombe
Homologous recombination
Conditional mutants
Suppressor mutants
Synthetic Lethality Screens
Drosophila melanogaster
Four pair of chromosomes, DNA double-strand
Eukaryotic
It can be easily transformed with transposable-element vectors
60% of its genes are also in the human genome
Study of several diseases (cancer, neurodegenerative diseases, blood disorders, immunological disorders)
Mouse
Genome just a bit smaller than human, but with quite the same number of genes
Disadvantages
: different in cognitive function, behaviour and gene expression; difficult to monitor development in utero; diploid vs haploid; a large genome that makes it harder to target and work with than organisms with more compact genomes
Inbred strain
Spontaneous mutations, induced mutations in known genes, addition of exogenous/modified genes, cell type-specific or temporal specific mutations, random mutagenesis, toxins, diet, radiation
Knock-out
Isolate the gene by PCR and then made a construction with neomycin as selection marker
Add an extra part that confers sensitivity to ganciclovir
Take stem cells from a brown mouse and introduce our DNA construction in it. We can have 3 situations: homologous recombination, random insertion and wild-type.
Take cells with homologous recombination and put them in a blastocyst, obtaining a
chimeric mouse
Tet-on/Tet-off system; lac system; cre-lox system
Plants
Transposons
Ac (Activator)
encode for a transposase;
Ds (Dissociation)
is an element that can jump but not by itself, because it needs for Ac
Used when you don't have efficient transformation
A
disadvantage
is that transposon is an unstable element, so it can move to another gene, and you lose your phenotype.
T-DNA insertion
T-DNA
is a plasmid that comes from bacteria and it induces tumours. It encodes for certain genes that cause production of components important for the bacteria growth. The genes are prokaryotic but the promoter is eukaryotic.
We can have 3 different possibilities: intron, exon or promoter
For each insertion you need a different plant
T-DNA with transposons
Putting Ac and Ds, where Ds can jump close to the T-DNA or far away. The inverted repeats of Ac have been removed
After the first generation, we take seeds and plant them again. On the T-DNA there is a negative selection marker SU1 (its expression makes plants sensitive to sulphuronyl urea, so plants that have T-DNA will die). On the Ds instead there is a positive selection marker BAR (provides resistance to the herbicide BASTA).
Double selection for only plants without T-DNA and with Ds.
Addition of a primer in the flanking regions and amplification by PCR
Pool different plants
PCR up the pools and see what hyper-pool is positive, and so on until we find our mutant
Check super-pools, pools and single mutant making an hierarchy job
Enhacer trap
: activation tag line contains a strong enhacer that when it goes near a gene, activate it and overexpress it and you can get very different phenotypes.
Gene trap
: fusion of the gene of interest with a reporter gene in frame.
Promoter trap