Genetic Engineering and Applications

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  • Created by: Sarah
  • Created on: 30-04-17 17:25
what happens in RNA inteference?
presence of double stranded RNA, recognized+cleaved by dicer -> give mircroRNAs -> microRNAs form part of RISC-> cleavage of mRNA by slicer -> cleaved mRNA is destroyed->no translation
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what does RISC stand for?
RNA induced silencing complex
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where is the protein of interest attached to in yeast-2-hybrid?
attached to DNA binding domain of transcription factor
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what do you use to find out what proteins regulate a certain protein of interest?
yeast 2 hybrid screen
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what is a yeast 2 hybrid screen looking at?
looking for proteins that physically interact with the protein of interest
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what happens in a yeast 2 hybrid?
POI on DNA Binding domain, lots of unknown proteins on AD activation domains, construct a POI plasmid+ a fish plasmid (plasmid with unknown pros on ADs) plput in yeast cells, selection in aa deficient media
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why in a yeast 2 hybrid do you put it in an amino acid deficient medium?
only those with both plasmids grow, the fish plasmid (unknown pro on AD) and POI plasmid
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what needs to interact for the gene to be expressed?
protein on BD and protein on AD to ineract
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how are the unknown proteins that bind to the POI identified?
by isolating yeast colonies, isolating plasmid containing cDNA for unknown protein and sequence it
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how do you find out the function of a certain protein?
observe absence by knockdown or knockout
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what comes under target validation?
1) knockdown using siRNA 2) Gene knockout in transgenic mice
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what does siRNA stand for?
short interferring RNA
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what does the RNA inteference pathway do?
refulate gene expression using microRNA
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one of the first thing in the microRNA inteference pathway is dicer what does it do?
cleaves double stranded DNA into 21-22 long fragment
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what happens when you experimentally do a siRNA gene knockdown?
foreign double stranded RNA is introduced into cells -> dicer cleaves -> risc degrades target mRNA
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2 methods to generate transgenic mice?
1) pronuclear injection 2) gene targetting
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in pronuclear injection where is foreign DNA (trangene) introduced into?
nucleus of fertilised ova
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how many copies of the transgene are inserted in pronuclear injection?
several
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where is DNA integrated in pronuclear injection?
at random sites in the genome
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what is pronuclear injection used to generate?
GM crops and animals
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why is each trangenic line in pronuclear injection unique?
each foreign DNA (transgene) can be modulated by the insertion site (variable expression patterns)
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what happens in gene targeting?
ES cells from mouse-> target vector electrocorporation->chose cell with target gene->grow in colony->inject ES cells into host blastocyst->implant in foster mother->give birth to chimeric mice->breed chimeric
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what 2 gametes are produced from chimeric mouse?
normal host blastocyst gametes and modified ES cell gametes
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how do you produce a PrP(protease resistant protein) Knockout mouse?
target the PRNP gene
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what is the difference between the homozygous and heterozygous protease resistant protein knockout mutant?
heterzygous have a longer incubation before onset of the gene
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what did trangenic mice with different levels of expression of prp confirm with pronuclear studies?
incubation time is inversely related to PrP expression
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where is foreign dna introduced into in gene targetting?
cultured mouse stem cells
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what is gene targeting used to generate?
knockins or knockoiuts
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where in the genome does gene targeting intergrate DNA?
specific sites in the genome
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what does microarrays use to hybiridse to each other?
single stranded DNA
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what happens to non-complementary dna in microarray hybridization?
it hybridises to other probe cells or its washed away
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what does red mean the gene has become in microarrays?
up regulated
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what does a microarray chip contain?
a grd of thousands (45,000) probe cells
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what do microarrays compare?
the transcribed genes
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2 applocations of genetic engineering?
clinical medicine, forensic science
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2 reasons for a genetic scree?
1) to predict 2) to diagnose
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what is a genetic screen used to predict example?
variants of BRCA1 and BRCA2 are associated with variable risk of developing breast cancer
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what do BRCA1 and BRCA2 do in breast and ovarian tissue?
control cell division
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what is a diagnostic example of a genetic screen?
detection of invading organisms (PCR to screen blood for HIV) or endogenous genetic mutations (SCA)
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what does REFLP stand for?
Restriction Fragment length polymorphism (variatios)
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what does it mean that a mutation may create a polymorphism?
a polymorphism in the number/sizes of DNA fragments produced by a particular restriction enzyme
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what can polymorphisms be usefil for?
a marker for disease as its a marker for the mutation
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Card 2

Front

what does RISC stand for?

Back

RNA induced silencing complex

Card 3

Front

where is the protein of interest attached to in yeast-2-hybrid?

Back

Preview of the front of card 3

Card 4

Front

what do you use to find out what proteins regulate a certain protein of interest?

Back

Preview of the front of card 4

Card 5

Front

what is a yeast 2 hybrid screen looking at?

Back

Preview of the front of card 5
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