Hi everyone! I will be the first in my group to share about what I have learnt during my first week of attachment. Attachment HERE is very fruitful because I get to learn from my mentor, we have a comfortable environment for work and the laboratory is very well equipped. =)
I am attached to a research laboratory which deals with delivery of proteins, genes and drugs into cells. The particular laboratory I am working in focuses on delivery of genes into mammalian cells, hence we are required to carry out a lot of Molecular Biology and Mammalian Cell techniques. My MP would also be on the delivery of certain genes into mammalian cells to get a certain desired outcome.
The delivery of genes is not as straight forward as I thought, because we have to keep constructing new viruses for each new type of gene we want to deliver! The virus vector is constructed to carry the desired gene so that it can be transferred to the mammalian cells via transduction.
For the whole of the first week, I have been learning how to construct viruses and how to measure the titer of the viruses that my mentor has already constructed. I am still learning how to construct the virus as it involves many steps. So far, I have only performed up to the digestion of the vector and the insert (step 6).
Outline on how to construct a Virus (The main points are in red, the details are for those who may be more interested in how the step goes about).
1) Culture the cells containing the desired gene that we want to package into the virus, on a tissue culture flask. Cells will adhere onto the treated surface of the flask, spread and grow. 2) Maintain the cells in healthy condition and prepare a suspension culture (by trypsinization) for counting and DNA extraction. Trypsin detaches the cells from the surface of the flask by breaking down protein that binds cells to the flask surface. Counting of cells is required as part of the DNA extraction protocol for maximal DNA extraction yield.
3) Use DNA extraction kit such as DNeasy® Tissue Kit to extract DNA from cell suspension and measure the concentration of DNA extracted.
4) Run the extracted DNA on agarose gelto confirm that the DNA is pure and check what is the kilo base pair of the DNA by comparing it with the DNA ladder marker.
5) Excise the band of DNA and dissolve gel to extract DNA using a gel extraction kit.
6) Obtain a suitable vector backbone to be ligated with the insert (gene) and perform restriction digestion and ligation.
7) Transform cells with recombinant vector and select out the cells via antibiotic resistance and blue white colonies screening strategy.
8) Extraction of recombinant DNA using mini-prep kit.
9) Send the constructed recombinant DNA for sequencing to confirm the gene of interest is in the correct orientation for expression.
10) Tranfection of cells (to be infected by virus) with recombinant DNA. Transfection is a process of transferring DNA into the cells that are to be infected by the virus. The DNA is complex with some salts (calcium phosphate) from the transfection reagent that facilitate transport into the cells.
11) Virus is used to infect the transfected cells, and viruses will package these genes into their capsid as they replicate.
12) The virus titer is measured using plaque assay. (pfu/ml).
13) If the titer of the virus constructed is too low, we would need to amplifythe virus.(another protocol). 14) Store these constructed virus stock in 4 degree Celsius cold room.
On the first day of my attachment, my mentor is already testing the efficiency of 2 types of viruses he has constructed months ago. He tested the efficiency of the viruses by measuring the virus titer (pfu/ml) using plaque assay. (Refer to below). Measuring the titer of the virus can be used for 2 purposes:
Determine the titer of virus constructed. To determine the titer of virus stock that has been stored for a long time.
He needs to do this because the viruses are stored in 40ml centrifuge tube in the cold room at 4 degree Celsius, and overtime the virus titer will drop from 1 x 10^7pfu/ml (initial) to as low as 2.5 x10^6pfu/ml after 3 to 4 months. Measuring the titer is very critical because if the virus titer is too low, it will not be effective in infecting the cells and gene delivering efficiency consequently will be low.
Outline on how to perform Plaque Assay in a 6 well plateformat
1) Harvest the cells that the virus can infect on the 6 well plates and incubate til cells are 50% confluence.
2) Perform serial dilution of the virus such as 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7 and 10^-8. in eppendorf tubes.
3) Remove medium from the cultured cells and add the diluted virus to the 6 wells and incubate to allow viruses to infect cells.
4) Remove the fluid (containing virus) in each of the well, as the viruses has already attached to the cells.
5) Add plaquing medium, which is a type of agarose gel containing medium to each of the well after the fluid is removed, and allow agarose gel to harden before incubating the plate of cells.
6) The function of the agarose gel is to immobilize the viruses so that it forms a clearing as it infects the cells.
7) These clearings (plaques) can be counted as plaque forming unit/ml, which is the titer of the virus.
That’s all for now! Thanks for reading my long entry! =)
