Transgenic Technology Flashcards
Describe the difference between forward and reverse genetics:
Phenotype driven - have a patient with a genetic disorder and phenotype abnormality and work backwards to determine the genetic mutation responsible
Reverse genetics - exact function of the gene is unknown, and the function of the gene is discovered by generating transgenics etc. in vitro or in vivo.
Describe the basics of model systems in transgenics:
Can be simple to complex, with increasing technical difficulty
The mouse model has been traditionally used for transgenic work
Now the zebrafish is being looked at because it is much simpler and easy to manipulate
Describe the generation of transgenic mice:
A reverse genetics method (gene is known)
Make the transgene construct with sequence gene and upstream promoter
Collect fertilised eggs from a mouse hormone induced mouse
Inject the transgene sequence into the male pronucleus, and inject into another mouse (hormonally treatment to accept the embryo)
Assess the genetics of offspring for the incorporation of the transgene (forming the founder transgenic line)
Describe transgene integration:
Transgene integrates in a ranom fashion (not targeted), meaning it may integrate into an area which can cause damage
Thus it is important to raise several founders to ensure the phenotype is attributable to the gene
Describe the generation of knock-out mice:
Needs to be a characterised locus (introns, exons, flanking DNA) to generate the KO vector/construct
Replace sequences with cassette (with neomycin resistance)
Add construct to ESCs and add neomycin - select for clones resistant which represent having an integrated vector
Clones are injected into a foster mouse
Look for chimeric offspring, which can be crossed to give the phenotype of the ESCs.
Describe gene targeting in mice:
Clones need to be tested to ensure they have replacement of the gene locus.
Homozygotes represent gene knock-outs
What is a conditional system:
Systems which can be used to turn off the gene when and where they want to
Describe the LoxP-Cre inducible system:
Bacteriophage derived - cre enzyme catalyses the formation of a lox loop, which is excised from the genome
The first mouse line has cre-recombinase somewhere and the promoter, and it is crossed with a second mouse line containing the targeted gene flanked by loxP
When crossed, these cause gene knockout
Excision only occurs in the tissue where cre-recombinase is expressed (where promoter is active), thus gives a lot more control
Describe what the loxP system has demonstrated:
All cell types of the pancreas come from endoderm expressing the TF Pdx1 (Pdx1 used as tissue specific promoter)
Describe the Gal-4 inducible system:
Derived from yeast to ectopically overexpress a transgene
When two transgene lines are mated, the offspring will show tissue-specific expression of the target gene, giving control over tissue specificity
The Gal-4 TF is an enhancer trap (weak promoter) and needs to integrate near the endogenous enhancer
Describe the inducible tet-off system:
In the presence of tatracycline, TetA (an activator of the tet operator) is sequestered and the target gene is inactive
When tetracycline is removed, the target is activated
Describe some experimental considerations of using genetically modified mice in research:
Integration effects which can have non-physiologic effects: Insertional mutatgenesis Epigenetic silencing Mosaicism Multi-site integration Sex chromosome integration
Different mouse strains may behave differently
In gene targeting, compensation can occur whereby knocking out or inhibiting the function of a gene can result in another gene taking over some of its activity
Name some systems available for cardiac-specific transgenesis:
Inducible:
CAG-CAT - permits conditional transgene expression in any tissue due to its ubiquitous promoter but is subjected to cardiac-specific gene/cDNA induction by the coexpression of a cardiac-specific Cre
MerCreMer - tamoxifen-inducible system used for more specific temporal control of expression as recombination events are dependent on the administration of tamoxifen
Describe the zebrafish as a model vertebrate:
Embryonic development external to female
Optical clarity
Development of body plan complete at 24 hours
High fecundity
Short generation time
Maintained at high density
Because their embryos have phenotypic similarly to human foetuses, they can be used to study developmental processes of some diseases
Describe morpholino knockdown of zebrafish:
Morpholinos interact with transcript translation
Allows for the analysis of gene function with a knocked down protein (as opposed to a KOs which may abolish viability)
However, there are some false positive drawbacks
