Transgenic + gene targeting Flashcards
Identifying a gene of interest
- 3 types of method
Transcriptome profiling
Protein-based methods
Whole genome sequencing
Transcriptome profiing
- what is it?
- examples
Looking at RNA expression in a particular cell type
(e.g. cancer cell line)
RT-PCR
- if you know gene effected
DNA microarrays
- see which genes bind to probes
RNA-seq
= turn all RNA into DNA
-> sequence using next gen. sequencing
(will show genes up or down regulated by drugs)
Protein-based methods
- examples
1- and 2-hybrid screening
= protein-protein and protein-DNA interaction tested by binding
Immunoprecipitation
= precipitating an antigen out of a solution using an Ab
Whole genome sequencing
GWAS
V cheap now
In vitro approaches
Cell + tissue 2D culture
3D tissue culture + organoids
In vitro limitations
Not all cell types can be cultured
Limited matrix + cell interactions
No recapitulation of forces
- hard to model intracellular forces
No org-level analysis
e.g. learning, stress response
Manipulating genes in vivo
- methods
Manipulation in bacteria
Transgenesis
Conventional gene targeting
Genome editing
Transgenesis
- allows..?
- typical transgender construct types
Visualisation of gene expression in a whole animal
- promoter + gene of interest + reporter (e.g. GFP)
- promoter + reporter
Transgenesis
- process
Pronuclear injection of tg DNA into 1 cell embryo
- > integrates into genome in quasi-random fashion
- > embryo gives rise to offspring
- > all cells in offspring have tg (due to mitosis)
Transgenesis
- efficiency
approx 10% of offspring are transgenic
ES cells
- uses
Take ES cells from ICM of 1 blastocyst
- > inject into another blastocyst
- > can contribute to development (pluripotent)
ES cell gene targeting method
- Make targeting vector DNA construct
- Either:
+ve selection (e.g. Neomycin resistance) for all TV integration events
Or
-ve selection (e.g. Thymidine kinase) for homologous integration events
Targeting vector
- other features
Homology arms each side of targeted insertion
- recombine by HDR (homology directed repair) with genome
ES cell gene targeting method
- selection
+vely select for Neomycin-resistant cells using G418
-vely select (lost gene product) for Ganciclovir-resistant cells
(normally Ganciclovir metabolised by Thymidine kinase to produce cytotoxin)
Screen to eliminate false +ves
Confirm using whole genome sequencing
Mutant ES cells –> mutant mice
- method
- Inject 8-10 targeted ES cells into blastocyst
- Transfer infected blastocyst into pseudopregnant mother
- ES cells integrate into development
- Offspring contains embryo cells + targeted ES cells
- ES cell presence revealed by coat colour
