What is the common method of cell transfection
The scientific research path is very long. Today we will look at the cell transfection experiment.
First, let's look at some common ways of cell transfection:
I have summarized several classical traditional methods,give you an introduction here.
1.Calcium phosphate co-precipitation
Principle: this method can be used for instantaneous or stable transfection.
However, because of its pH, temperature, and sensitivity to small changes of buffer salt concentration, the result it's easy to have a difference, and of many types of cell cultures (especially primary cell) with cell toxicity, low transfection efficiency.
Experimental steps: mixing nucleic acid and calcium chloride in phosphate buffer, at the same time control the condition such as pH, temperature and room temperature incubation, generate the tiny insoluble particles condensed DNA precipitation to scatter particles type precipitation into cells, promote DNA adhesion on the cell surface, coprecipitation by analyzing the swallowing function into the cytoplasm and in cell transient stability of gene expression or infection.
2.Artificial liposomes method
Principle: the phosphite group with positively charged liposomes and nucleic acid band negatively charged to form a compound, which can then be stabilized by the cells and transfected with the instantaneous time.
This method is applicable to almost all cells, with high transfection efficiency and good repeatability, but when transfection, the serum is removed and the transfection effect changes with the cell type.
Experimental steps: in a separate test tube dilution nucleic acid respectively and the liposome transfection reagent and combined with nucleic acid phosphate skeleton, form the positive charge on the compound liposome and help compounds with cell membrane to compounds through swallowing function within into the cytoplasm, or silence gene expression, transient analysis of cells.
3.Virus transfection
Principle: for cells that are not transfected with liposomes, virus transfection can be used.
Can be used in protein overexpression or inhibition, is the most commonly used method in clinical research.
It can be used to integrate the exogenous gene into the chromosome, which can be used to transfect the stability of the cell and the original cell.
Experimental steps: by gene clone, producing recombinant virus - by using a virus transfection packaging lines, amplification and recombinant virus particles were isolated and purified and titration poison disease - cell transduction purpose (contain virus specific receptors) to remove the virus in the culture, and add fresh medium - or silence gene expression, transient analysis of cells.
4.Electroporation method
Principle: use electrical pulses to form a temporary hole in the cell membrane to allow nucleic acid material to enter the cell through the hole.
Experimental steps: will punch in the buffer cells suspended in points, the preparation of cells - in the presence of a specific buffer, impose appropriate electrical impulses to electrical impulses on cells on the membrane potential difference formation, temporary hole formation, restoring the nucleic acid into the cell to the cell growth state, recovery and gene expression or silence, transient analysis of cells.
Advantages and disadvantages:
So what is the reason for the low cell transfection rate in the experiment?
I guess you don't know. Now let's see!
Factors affecting the transfection test:
1.The transfection agent does not match the cell line
Transfection reagents and cell lines are also well-coordinated, using the same reagent, and the efficiency of transfection of different cell lines is usually different.
However, the selection of cell lines is usually based on the needs of the experiment, so the suitable transfection agents should be selected according to the experimental requirements and the cell characteristics before transfection experiments.
Each transfection reagent will provide a list of cell lines that have been successfully transfected and the literature to choose the reagent that is best suited to the experimental design.
Of course, the most suitable is efficient, low toxicity, convenient and cheap transfection reagent.
2.Cell state change
Because some cell lines are unstable, different selection pressures may result from different selection pressures as the cultivation time changes and the conditions of cultivation are different.
So even in the same cell line, the difference in transfection ability under different conditions can be significant
(1) the transfection agent does not match the cell
Cell transfection is best suited not to the original cell, but also to the cells of many times.
This is because cell culture has evolved in the lab for months and years, with mutations, total chromosome recombination or changes in gene regulation.
This results in changes in the behavior of the cells associated with transfection.
The cells that are most suitable for transfection are the cells that reach logarithmic growth after several generations. The cells are flourishing and transfected most easily.
(2) seize the moment
That's right!
The transfection also has the right timing to compare undivided cells, which tend to ingest and express exogenous DNA more easily than still cells.
So for most transfection operations, cells are on the same day or the day before.
Equally important is the cell while the kind of plate for transfection should not be in the condition of excessive growth, such as too many number of cancer cells, each other, the depletion of nutrients, metabolic waste accumulation, low transfection rate also is very normal!
Therefore, it is necessary to transfect the optimal cell density to obtain the higher transfection rate.
Different transfection agents require that the optimal cell density of transfection is different, even if the same reagent is different depending on cell type or application.
(3) microbial disturbance
Cultures can be contaminated by bacteria, yeast, fungi, viruses, mycoplasma, and even other cell types.
All kinds of pollution lead to wrong results.
(4) cross-contamination
If different types of cells are cultured at the same time in the same laboratory, the phenomenon of "cell serial gate" can be agreed upon, causing cross-contamination
3.Transfection method
Different transfection agents have different transfection methods, but most of them are similar.
Transfection should be according to the specific transfection reagent recommended method, but also should pay attention to, because of different nature different laboratory cultures of cells, plasmid quantitative differences, the differences of operation methods, etc., its transfection effect may be different, according to specific conditions to determine the optimal transfection conditions of laboratory.
(1) serum
After transfection does not add the serum in time, can cause cell mass death.
In general, the liquid should be changed for 4-6 hours after transfection and to be replaced with a serum culture medium.
You can also add serum to the original serum without the serum.
At this time, it is better not to change liquid, do not disturb the cell, let it rest quietly.
But you can't add the serum too soon.
Premature, can cause the cell that does not transfection is crazy to grow.
So what is the best time?
When 20% of the cells are round, it is the best time to add serum.
But special attention should be paid to: serum is an additive that contains the inexact components of growth factors and other auxiliary factors, and there are significant differences in the growth of different cells.
Changes in serum quality directly affect cell growth, and therefore the transfection efficiency.
The preheating of the new medium is helpful for cell transfection.
(2) DNA quality
DNA quality is very important for transfection efficiency.
General transfection technologies such as liposomes, etc.), on the basis of the principle of charge attract if DNA is not pure, such as with a small amount of salt ions, proteins, metabolites in the formation of the pollution are significantly affect the transfection complex and transfection.
4.Carrier to build
The construction of transfection vectors (viral vectors, plasmid DNA, RNA, PCR products, oligonucleotides, etc.) also affected the transfection results.
Therefore, it is important to choose composition or regulation, and the proper promoter of strength is also important. Meanwhile, the transfection of the same carrier constructed by the carrier and other genes can be controlled to eliminate the interference of toxicity.
Therefore, the quantity of the transfected plasmid should be guaranteed to be more than 2 mu g.
The purity of plasmid is not sufficient or contains bacteria LPS or other substances that are toxic to cells, which may also affect the transfection efficiency.
At this time, the plasmid should be purified and concentrated.
These are the main reasons for low cell transfection rate.
prev:Is there a "gene transfer" in the history of human life? What is the level of synthetic life technology?(2017-08-04)
next:How to make a combo diagram?(2017-08-07)