Mammalian genome modification techniques Flashcards
(31 cards)
Ways of identifying a gene of interest?
Transcriptome profiling, protein-based methods, whole genome sequencing, mutation-based
Ways of doing transcriptome profiling?
targeted: RTase-polymerase chain reaction (RT-PCR)
DNA microarrays
RNA-sequencing (RNA-seq) (becoming default)
Benefits of RNA-sequencing (RNA-seq)?
V powerful–> can use reverse transcriptase to make libraries
Works well on single cells
Can see if proteins are expressed in a particular cell type/line
Protein based methods of identifying a gene of interest?
one- and two-hybrid screening
co-immunoprecipitation (IP
Protein profiling
What does ChIP stand for?
chromatin IP
How does co-immunoprecipitation work?
antibody against protein of interest is used to pull down the protein, and can also pull down any other proteins linked looks for interactions
Ways of doing whole genome sequencing?
genome-wide association studies (GWAS)
Mutation based ways of identifying a gene of interest?
Induced
naturally occuring
Induced ways of doing mutation based ID of a gene of interest?
Forward and reverse screening
Objective of manipulating genes in vivo?
introduce a desired change to the genome
of all cells in an individual
What does manipulating genes in vivo usually first involve?
Manipulation in bacterial–> gene cloning
3 ways of manipulating a gene in vivo?
transgenesis
conventional gene targeting
genome editing
What is transgenesis?
integration of exogenous DNA so that it is part
of the genome, nominally of every cell in an organism
What does transgenesis allow visualisation of?
Gene expression in whole animal as it is in every cell
What is used to look for where the transgene is active?
A marker that is with the transgene
Examples of markers used to mark transgenes?
GFP, mCherry (red)
Basic idea of making a transgene?
generate a construct and cause it to integrate into the genome
integration occurs early in development so all the cells contain it
What is pronuclear mincroinjection?
inject transgene (tg) DNA into
the pronucleus of a one-cell embryo
What is ICSI transgenesis?
microinject sperm plus tg DNA into
unfertilised oocyte (ICSI)
When does the transgene integrate into the genome in pronuclear microinjection and ICSI transgenesis?
B4 or during the 1st S phase
Where does the transgene integrate into the genome in pronuclear microinjection and ICSI transgenesis?
Quasi random
What is done with the embryo after it has the transgene injected?
Transferred to the mother
How many transgene integrants do cells in the offspring of the one injected with the transgene have?
1-100, usually 1-5
What is the result of less than 100% of the cells having the transgene?
Animal is mosaic
