Lab report 3: Cell storage, cell migration assay and gene manipulation This sect
ID: 69954 • Letter: L
Question
Lab report 3: Cell storage, cell migration assay and gene manipulation
This section includes 1) Cell storage process; 2) Study cell migration using CFA; 3) gene transfection.
Purpose and principle of each experiment:
Cell storage:
Colony Forming Assay
DNA transfection:
Methods and protocols of each experiment:
Cell storage: list the temperature of from high to low during cell storage process. Write down the step-wise protocol for cryopreservation:
CFA approach:
Protocols for DNA transfection in cells:
Results and discussion:
Why are cells maintained at low temperature? What can cause cell death during storage (discuss those factors involved)? Why adopt a gradual temperature strategy? How to improve cell storage quality? Give example where cell storage is applied.
What are your results of the CFA? Give examples where CFA technique can be applied.
What are the results of your transfection? What is the ratio of your transfection? How to improve transfection ratio? (cell lines, DNA, lipofectamine etc). What are the best conditions for transfection?
Discuss how to achieve gene overexpression, down-regulate gene function and totally disrupt gene function (what technique you need to use). Discuss the properties of vectors.
What is the application of gene manipulation and what can be done with this?
Explanation / Answer
Purpose and principle of each experiment:
Cell storage:
To maintain cells in culture without allowing them to overgrow.
The cells were stored to obtain them in increased number for experiments or further storage
Colony Forming Assay
To study the ability of a cell to proliferate to form a colony or a clone.
It also helps us to study the effectiveness of specific agents on the survival and proliferation of cells used in the culture.
Finds application in tumor science, where the effect of drugs or radiation on proliferation of tumor cells can be evaluated.
DNA transfection:
The aim is the insertion of genetic material either DNA or double stranded RNA into the cells.
Through DNA insertion we can achieve the expression and production of proteins in the cell. Whereas the RNA insertion can down regulate the production of specific selected protein by stopping the mechanism of translation.
Methods and protocols of each experiment:
Cell storage: list the temperature of from high to low during cell storage process.
For storage, cells are cooled from 0 ºC to -20 ºC.
To stop biological time, the cells will be maintained at -130
To store for years, the temperature is -70 ºC to -90 ºC.
In electric freezer, a temperature of -135 ºC can be maintained.
With liquid nitrogen we can achieve -196 ºC.
Write down the step-wise protocol for cryopreservation:
1. Supend the cells in growth medium at room temperature (concentration is 2x106 cells per ml).
2. Add cryoprotectants (Glycerol or DMSO of 10% conc) to the growth medium to avoid damage during freezing.
3. Mix the cells with cryoprotectants bringing the final concentration of cells to 106 to 107 cells per ml with 10% cryoprotectant and leave them for 30 minutes to allow the protectant to enter into the cell.
4. Now take 1ml aliquots of the prepared cell suspension in cryotubes and cool the temperature 1 ºC per minute till it reaches -50 ºC and then lower it to -140 ºC to -196 ºC depending on the requirement
5. Store them in vapor phase of liquid nitrogen maintained at 140.
CFA approach:
Prepare the required dilution of cells needed for plating.
Try to prepare the suitable media by autoclaving following by plating.
The specific volume of diluted cell suspension is poured on the media and is incubated at 37c for 24 hours.
Visualize the colonies under a microscope and count the number of colonies.
Protocols for DNA transfection in cells:
Take the log phase cells to maximize the transfection efficiency
Harvest the cells the day before and count the cell and store them in a multi well plates at a concentration where it will have correct level of confluency
Now add the chemical agents and nucleic acid in a separate tube to form nucleic acid and reagent complexes based on the type of cell we want to transfect.
Now add this mixture to the cells need to be transfected and leave for overnight.
The nucleic acid and reagent complexes attaches to the cell and mediate transfection
To check the completion of transfection, remove the cells and plate on a suitable media
Results and discussion:
Why are cells maintained at low temperature? What can cause cell death during storage (discuss those factors involved)? Why adopt a gradual temperature strategy? How to improve cell storage quality? Give example where cell storage is applied.
At low temperature, we can arrest all the physiological activities and growth of the cell.
Cells die during storage because of the formation of ice crystals outside and which penetrate into the cell or sometimes tiny crystals are also formed inside the cells.
We need to reduce the temperature gradually like 1C per minute till it reaches 50C to avoid any type of osmotic shock to the cells due to immediate decrease in temperature.
By using correct concentration of cells, by mixing them with required cryoprotectants and storing them at desired temperature we can improve cell storage ability.
Life cell umbilical cord preservation is helpful to save life whenever a need will arise. Storing of different cells are useful for research studies. Preservation of novel bacterial or other microorganism cells are useful for future applications. Whenever needed, these cells can thawed and cultured. No need to go for isolation.
What are your results of the CFA? Give examples where CFA technique can be applied.
CFA allows the study of response of malignant cells to the treatment condition
To study the cell morphology and their colony forming format
The CFA can be used to assay for new drug screening.
What are the results of your transfection? What is the ratio of your transfection? How to improve transfection ratio? (cell lines, DNA, lipofectamine etc). What are the best conditions for transfection?
By using proper transfection reagents.
Use enough plasmid DNA along with nuclear DNA
Maintain optimal cell density
Maintain proper DNA:transfection ratio
Make sure inhibitors should not be there in the mixture
Cells should not undergo any change
Maintain proper temperature and sufficient time for the reaction to occur
Optimal cell concentration and cell viability, properly maintained transfecting agents, correct size of the plasmid DNA can improve transfection efficiency.
Discuss how to achieve gene overexpression, down-regulate gene function and totally disrupt gene function (what technique you need to use). Discuss the properties of vectors.
What is the application of gene manipulation and what can be done with this?
With gene manipulation, we can make any cell to synthesize the protein of our interest. The cells can be made to synthesize, enzymes like insulin, hormones like growth hormones, vaccines, antibodies, and many drugs. Finds application in research, agriculture. Can be used to find the function of any unknown genes. Through gene manipulations, we can construct bioreactors for the production of enzymes and other products.
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