Genome Editing and Sophisticated Transgenesis Flashcards
(34 cards)
what is transgenics?
introducing foreign DNA into an organism’s genome
- can study the function of genes by permanently adding new/modified genes = passed onto offspring
what is random transgenics (using pronuclear injection)?
foreign DNA integrates into a random location in the host organism’s genome - no specific targeting
how is random transgenesis performed?
pronuclear injection
- DNA injected into the male pronucleus of a fertilized egg (zygote)
- injected DNA integrates randomly into the genome at various locations - no control over the insertion site
- embryos are implanted into pseudo-pregnant females to develop
- offspring are screened for transgene integration
What is a promoter trap and how does it work?
promoter trap = promoter-less reporters relying on host gene activity to express
promoter-less (promoter trap) constructs have a reporter gene, splice acceptor site (5’ end) and NO PROMOTER (can’t drive own expression)
random integration of trap into genome - exp. depends on insertion site
- lands inside actively transcribed gene = host’s transcription machinery splices into the trap
- host gene’s promoter drives expression of the reporter
result:
- reporter only turns on if the trap inserts into an active gene
- good for identifying active genes and studying enhancer activity
- use antibiotic resistance genes (like neo) linked to the reporter to select only successfully integrated cells.
What is the Rosa26 mouse model and why is it important?
Rosa26 locus = genomic site supporting ubiquitous transgene expression
- inserting reporter genes (e.g. GFP) - live tracking of gene exp. in all tissues
- allows lineage tracing & studying widespread/tissue-specific gene activity
How is the BAT-gal mouse model used to study Wnt signalling?
BAT-gal mouse carries Wnt-responsive TCF/LEF sites driving β-galactosidase expression
- can visualise in vivo Wnt pathway activation during development
- can study tissue-specific and developmental responses to Wnt signalling
what is targeted transgenesis using homologous recombination?
precise change in the genome - foreign DNA is integrated into a specific location in the genome
how is targeted transgenesis performed using homologous recombination?
homologous recombination = exchange (recombination) of genetic information between two similar (homologous) strands of DNA
- introducing a DNA construct with flanking homology arms matching the target gene
- conducted in embryonic stem cells = cell’s repair machinery uses these arms to swap the genomic DNA with the construct = precise gene editing
- successfully modified ES cells are selected and implanted into embryos = produce transgenic offspring
What do the Tet-On and Tet-Off systems allow researchers to do?
allow controllable expression of a transgene using doxycycline or tetracycline as a switch
What is the Tet-On system and how does it regulate gene expression?
Tet-On system uses reverse TetR (rtTA), a modified version of the Tet repressor, to activate gene expression
presence of doxycycline
- rtTA binds to TRE (Tet-response element) = activates transcription
no doxycycline
- no rtTA binding to TRE = no gene expression
What is the Tet-Off® system and how does it regulate gene expression?
Tet-Off system uses the Tet-controlled transcriptional activator (tTA), which is a fusion of TetR (Tet repressor) and the VP16 activation domain.
no doxycycline = tTA binds to the TRE = activates transcription
with doxycycline = tTA can no longer bind to TRE = gene expression OFF
How does the Tet-Off system utilize doxycycline in gene regulation?
Tet-off system doesn’t need doxycycline for gene activation - doxycycline prevents tTA from binding to TRE = turns off gene activation
How does the Tet-On system utilize doxycycline in gene regulation?
Tet-on system needs doxycycline for gene activation - with doxycycline, rtTA binds to TRE = activates gene expression
How does the Tet system allow for precise control of gene expression in transgenic organisms?
Tet-On and Tet-Off systems allow precise control over gene expression by using doxycycline to turn the gene ON or OFF
can be used for studying genes in specific tissues/ time points without permanently altering the gene’s activity - ideal for inducible gene expression
Why is the oestrogen receptor (ER) pathway useful for controllable gene expression?
allows for inducible gene expression using oestrogen or tamoxifen as triggers
by modifying the ER system, it only activates when the ligand (e.g., tamoxifen) is added not affected by endogenous oestrogen)- temporal control over gene activation
examples of a method for inducible gene expression
- Tet-on/ Tet-off system & doxycycline
- oestrogen receptors responding to other oestrogen-responsive proteins
How does CRISPR-Cas9 work in targeted transgenesis for genome editing?
- guide RNA directs Cas9 enzyme to a specific DNA sequence
- Cas9 enzyme creates a dsDNA break
- break repaired by cell’s repair machinery - either leads to:
1) knockout via NHEJ (error-prone repair)
2) introduce specific edits via HDR (homology-directed repair, if a template is provided)
CRISPR is faster and easier than homologous recombination - more efficient for gene modification
In targeted transgenics, how do we select successfully modified embryonic SCs using homologous recombination? (positive and negative selection)
positive selection
- insert resistance gene (e.g., neo, conferring G418 resistance) into recombined region to select for correctly modified cells
negative selection
- place sensitivity gene (e.g. tk (sensitivity to ganciclovir)) outside homology region
- cells with random integration incorporate tk and are killed
- cells with proper homologous recombination survive both selections
cells that survive positive selection = have integrates resistance gene - successful
cells that don’t have sensitivity gene = no sensitivity gene - successful
What are the main applications of CRISPR-Cas9 in transgenesis?
gene knockouts
gene editing - precise mutations
disease modelling (in animals)
gene therapy - correcting genetic disorders
How can gene expression be silenced using transgenics? (shRNA & antisense RNA)
shRNA - sequence introduced, forms a hairpin-structure, leads to degradation of the target mRNA = silences gene expression
antisense RNA - complementary RNA that binds to target mRNA = prevents translation = gene silenced
What is the role of reporter genes in transgenic studies?
e.g. GFP, beta-galactosidase
- track gene expression
- linked to regulatory regions (promoters) of the gene of interest = can visualise gene activity
GFP = live imaging of gene expression in living tissues
β-galactosidase = colorimetric readout using X-gal to track gene activity
What is a conditional knockout mouse model?
allows for the specific deletion of a gene in certain tissues or at certain times
- uses Cre recombinase to perform loxP recombination
- causes gene knockout only when Cre is activated
How does the Cre-Lox system work in transgenic animals to create a conditional knockout?
Cre-Lox system uses;
- Cre recombinase = enzyme that recognizes loxP sites flanking a gene
Cre is expressed - cuts out DNA (GOI) between loxP sites (floxed gene)
- allows conditional gene knockout = gene is deleted in specific tissues/ at specific times/ in response to certain external factors (e.g. drug)
e.g. Cre-ERT = Cre recombinase under control of tamoxifen - in presence of tamoxifen = Cre activated = excises GOI
by placing Loxp sites around DNA (‘floxing’ gene) - enables spatiotemporal regulation of gene activity
How does the Cre-ERT (oestrogen receptor-tamoxifen) system work for time-controlled gene deletion?
Cre-ERT system uses a modified oestrogen receptor ligand-binding domain (ERT)
- no tamoxifen = Cre-ERT fusion proteins remains inactive
- with tamoxifen = Cre-ERT translocates to nucleus = excises gene between LoxP sites = gene deletion/conditional knockout at a specific time
