AMB10 Flashcards
(22 cards)
describe forward genetics
create and isolate mutants in which phenotype is altered, identify mutations in a given gene.
describe reverse genetics
genes are isolated and the functional link to phenotype is made by creating mutations and observing their effects on the phenotype
what is transgenesis
process of adding an exogenus gene into an organism
what are the type of transgenesis
gene ablation- complete abolition of gene down reg over expression mutation mis expression
What are the methods used to introduce DNA into a cell
artificially -microinjection, -PEG/Cal2, -biolistics, -electroporation Naturally - bacteria - viruses (transgenesis happens all the time in nature)
Describe microinjection
physically inject,
works for big cells, just one cell at a time
relies on cells own repair mechanisms to integrate DNA
Describe the three steps of making transgenic mice
- microinject gene of interest as linear DNA into male pronucleus
- introduce several such embryos into pseudopregnant mouse
- somatic cells of offspring tested for presence of gene after biopsy and selected mouse bred to test for gene in germline.
describe biolistics
use a particle gun to introduce DNA into tissues,
DNA coated onto microscopic metal spheres (gold/tungsten) and then literally shot into cells by sudden release of high pressure helium jet
common for introduction into plant tissue
describe electoporation
commonly used for intro of DNA, RNA into microbes or single celled organisms
cells bathed in solution containing DNA, then subjected to high voltage which is though to create temporary pores in the membrane so DNA is taken up by the cell and integrated into genome.
describe the steps of using agrobacteria to transfer genes into tobacco plant
- discs removed from tobacco leaf
- leaf discs incubated with genetically engineered Agrobacteria for 24hrs
- selection medium only allows plant cells that have acquired DNA from bacteria to proliferate
- moved into a shoot inducing medium
- transfer shoot to root inducing medium
- grow up rooted seedling
- adult plant carrying transgene that was original present in bacteria
How is Agrobacteria tumafaciens used to transfer DNA
contains Ti tumour inducing plasmid, T-DNA which can be incorperated into the nucleus by the hostss own machinery.
- usually produce hormones causing tumour to form and cells proliferate out of control and produce lots of sugars for the bacteria to feed of of.
- Ti plasmid can be used to carry genes.
- adaradopsis flowers can be dipped in Agrobacterium solution and Ti plasmid can enter intoo flowers and seeds = fast and efficient
What types of genome editing can be achieved
change gene expression using a strong constituitive promoter to over express
conditional expression with inducible promoters
silencing using antisenseRNA (asRNA)/ RNAi
modified by deletion or knock out.
What can be used in gene targetting by homologous recombination
Ku mutants
Zn finger endonucleases
TALES and CRIPRs
What are TALEs
transcription activator like effectors
proteins found in the plant pathogenic bacterium Xanthomonas
What do TALEs do?
bind to promoter sequences in plant genes and activate expression of genes which aid bacteria infection.
what are TALEs made up of?
reppeats that bind DNA, sequence of repeats determine which DNA they bind
can be engineered to recognise any sequence wanted.
How can TALEs be used?
by associating TALE domain with nucleases or transcription activator complexes specific genes can be targeted.
give an example of a nuclease being fused to a TALE
fok1 - dna hydrolase
acts as a dimer to cut DNA
what is CRISPR
clustered regularly interspersed short palindromic repeats
= bacterial immune system
segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of “spacer DNA” from previous exposures to a bacterial virus or plasmid
What is \Cas9
DNA endonuclease found in many bacteria that functions as part of the defence system
contains 2 active sites that each cleave one strand of dsDNA - guided to target DNA by RNA that contains sequences matching sequence to be cleaved.
what does RNA guided cas9 activity create?
site specific dsDNA breaks which are then repaired by either non homologous end joiining or homolgous recombination
how can CRISPR be used to engineer genomes
by delivering engineered cas9 into genome can cut at desired sequence. during homologous recmbinatio addition of donor dna enables new sequece to be inserted at the break site
