Functional studies of gene variants Flashcards
2 strategies to express gain of function
- over-expressing the mutant protein
- Keeping allelic balance as it is a dominant transmission
What model is used to study gain-of-function?
Knock-in model: transgenic mouse
Which models are used to study loss-of function?
Knockout and knockdown gene expressions
What is knockout gene expression?
You get rid of the gene–> eliminate its expression
What is knockdown gene expression?
downregulating the gene’s expression significantly
What should not be modelled using a knockout gene?
Dominant negative mutation
Cell models that can be used
- Immortalised cell lines
- Primary cells isolated from the tissue of interest
- Induced pluripotent stem cells
- Organoids
What are organoids?
3d structures that resemble the organ of origin that are derived from stem cells which are then differentiated
RNA interference
KNOCK DOWN: a technique that uses siRNAs to target a specific RNA and silence its expression
Describe the process of RNA interference
SiRNA binds to complementary sequences of target mRNA which triggers the degradation of mRNA or the inhibition of its translation into a protein
How can a cell repair a double-strand break caused by CRISPR/cas9 knockout?
- Homology directed repair
- Non-homologous end joining NHEJ
Homology directed repair
Uses homologous recombination to synthesise the missing piece so that the precise changes or insertions can be introduced at the site of the break
NHEJ
error prone: features giving together the 2 strands of DNA without a template
What does gene targeting use?
Homologous recombination
Process of gene targeting
- Isolate embryonic stem cells from blastocysts and we can culture them in vitro
- The cells are placed in the presence of a targeting vector
- The plasmid will match and induce homologous recombination between the target sequence and plasmid causing an exchange of genetic information
- Cells that were successfully modified are injected into a blastocyst to create a chimeric blastocyst creating chimeric offspring
- The chimeric mouse is crossed with a wt mouse to obtain a heterozygous mutant mouse
Chimeric offspring
An organism containing cells derived from 2 or more genetically identical zygotes
What is used for positive selection?
Neomycin an antibiotic
What is used for negative selection?
Thymidine kinase
When we give ganciclovar to the cells, only the cells that express Tk will die
What is conditional inactivation?
Knock out a gene present in certain tissues
loxP system
- the target gene we want to eliminate is flanked with loxP sites: when recombination is induced, the gene between the 2 sites will be cut out
- The mouse is crossed with another transgenic mouse in which a tissue-specific promoter linked to a Cre gene is introduced
- The complex induces recombination between the 2 loxP sites which will be present in all cells of the body but the Cre enzyme will be present only in certain cells
- Their offspring will have the Cre transgene allowing the target gene to be eliminated in those specific tissues
What is another technique with similar principle as Cre?
Flippase (FRT)
What does it mean when we have the presence of the enzyme Cre?
it induces recombination
Inducible inactivation
Deletion of a gene only in certain tissues at a certain time
What is the principle of inducible inactivation?
Estrogen receptors are present in a cell in the inactive state and they are bound to HSP90, a chaperone that keeps the protein in the cell inactive. When we give estrogen to cells they bind to the estrogen receptor and upon this binding HSP90 releases this receptor.
What are the 2 methods to make Cre inactive?
- Fusing the Cre protein with a part of the estrogen receptor
- Tetracyclin resistance operon
Fusing the Cre protein with a part of the estrogen receptor
Estrogen receptor has been modified to bind an estrogen-like molecule instead of estrogen. Tamoxifen causes the release of HSP90 from teh estrogen receptor so the Cre protein becomes active. Cre will go to the nucleus and catalyse the recombination
Tet-off system
Tetracyclin/ Dox is sued to switch off the expression of the gene driven by Cre and activated by TTA protein
Tet-on system
rTTA is designed to let the system work in the other direction: when we give Dox cyclin, it binds to rTTA which binds to tetO and activates expression of CRe
What happens if you use CRISPR cas9 at the 2-cell or 4-cell stage?
Then part of the cells be edited and the others will be wt creating a mosaic
Transduction
Using a lentiviral/adenoviral vector to stably introduce the expression of a gene of interest within a cell
Transfection
Using an expression vector that contains a cDNA of the gene that we are interested in: can involve a nucleic acid sequence such as siRNA/ miRNA or a plasmid
What happens if a nucleic acid sequence is used in transfection?
The sequence will remain at the cytosolic level
What happens if a plasmid is used in transfection?
It may be introduced in the nucleus and integration within the DNA. Antibiotics are given so that only the cells with plasma integrated within their DNA will survive
Stable transfection
Transfection selected for integration
Transient transfection
Acute phase [short period of time]
Stable transfection vs transient transfection
- DNA vectors are used only; DNA vectors and RNA can be used
- integrates in the genome; not integrated in the genome
- passed onto progeny; not passed onto progeny
Hemizygous
has one chromosome carrying the transgene and one chromosome not carrying the trans gene
What should our control mouse be if we have overexpression of a mutant?
Our control mouse should have overexpression of out wt protein so that the overall level of expression can be comparable
What can avoid the trans gene from the regulation of chromatin?
Insertion of insulator sequences
