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What is a downside of using a knockout to study the function of a gene?
we cannot necessarily contribute the loss of function to the gene of interest because the other factors are not controlled for
What are other ways for studying the function of a gene?
deleting the regulatory region, which would no longer act on the gene of interest
We can delete the gene so it is no longer functional
We can also increase the activity with a constitutive promoter to get a gain/ increase of function
In cells, how can we study the function of a gene?
- we can employ certain tools to manipulate genes for loss or gain of function in whole organisms
- --> we can look at in vitro systems and get the cells to grow in the presence of growth factors, vitamins, sugars, carbon sources, etc.
What is a downside to in vitro studies of genes?
We cannot see the whole picture; it is different than having a whole organism
Why is studying the function of a gene in an organism better?
because it's a better model of the physiological model
What are the downsides with studying genes in organisms?
- Ethical problems
- Findings you get in an animal do not necessarily hold true in human models
Explain the structure of VASP.
- There are five phosphorylation sites on VASP
- - one is serine 239; PKG has a higher affinity for this
- by acting on this site, you can turn the function of VASP on and off
One way we can ask questions about genes is by ....___.
Example: VASP. Explain
Using drugs, which do certain things to the cell
We can treat a cell with a toxin to get results. ST toxin can be used--> phosphorylation of GC C-->cGMP-->PKG-->pVASP
What do invadopodia do?
They secrete proteases to degrade the membrane
What is the problem with concluding that GCC and cGMP can inhibit actin polymerization through the ST toxin pathway?
GCC and cGMP have several pathways. VASP is not the only pathway.
How can we deal with this?
We need to add genetic controls
How can we add genetic controls?
One way is site-directed mutagenesis
Explain site directed mutagenesis.
Mutant strand synthesis: perform thermal cycling to denature the DNA template, anneal mutagenic primers containing desired mutations, extend and incorporate primers with high-fidelity DNA polymerase
DpnI Digestion of Template: Digest parentla methylated and hemimethylated DNA with DpnI
Transformation: Transform mutated molecule into competent cells for nick repair
What does site directed mutagenesis allow us to do?
Allows us to alter the nucleotide sequence of VASP and introduces a mutation at site 239 that does not code for serine, but rather an amino acid that won't get phosphorylated
For VASP, it'll make the cell less metastatic
In your own words, describe site-directed mutagenesis.
- 1) Introduce mutation through the primers
- 2) Degrade methylated DNA because there is no methylase in PCR mix, meaning the new plasmids don't get methylated
- 3) Now that copies of the genes are made, put them in bacteria and grow to get alot of it
- 4) Put it in a cell and get expression. Dominant negatives are made, which are phenotypes with a loss of function)
Phenotype with a loss of function will be the dominant one. So, anything with A will be dominant. Ex: AA or Aa
this is a dominant negative because its the one most often expressed, but it causes a loss of function
Explain what a dominant negative for VASP will do?
Because it forms a tetramer, you lose endogenous function because the mutant and wild type may form the tetramer togeter
Explain what a mammalian expression vector is
MCS: Multiple cloning sites (entry point of the gene)
promoter- CMV viral promoter, which is a constitutive promoter in mammalian cells
AMP resistant gene
Origin for the start of synthesis
Neomycin resistant gene (neomycin kills mammalian genes)
What is the deal with neomycin?
Neomycin kills mammalian genes. So, any cells that don't have the neomycin resistant gene in their plasmid will die
How can we put the plasmid into mammalian cells?
- There are two ways:
- 1) transfection
PLasmid and cells are mixed with a transfection reagent
DNA liposome complex forms micelles with DNA packaged inside
Endocytosis of the micelles
Lysosome with acid degrades liposome but keeps DNA
**We need to get it into the nucleus. This will happen when replication occurs and the nuclear membrane is broken down.**
We have a set number of plasmids in teh system; and, since the cell number is increasing, the plasmids are being diluted. This leads to a burst of expression and an eventual fading away
a retroviral genome split between two vectors
a virus tranduces the nucleic acids into the bacteria