Which transfection reagent is better for 293T cells

Many eukaryotic expression vectors, such as those containing the SV40 virus replication start site, can replicate in cell lines expressing the SV40 virus T antigen, thereby increasing the expression level of foreign genes. 293T cells are a cell line expressing SV40 virus T antigen, which is ** human embryonic kidney cell 293HEK, and the T antigen is inserted into it after modification, so transfection with 293T cells can obtain higher expression levels. 293T cells can also be used as common mammalian cells to screen stably expressed cell lines, but they are not conducive to screening due to poor adherence and easy shedding.

3T3 cells are mouse embryonic fibroblast cell lines that have significant contact inhibition, making it easy to identify transformed cells.

293T is a human renal epithelial cell, and I personally think that it is difficult to transfect; 3T3 is a fat cell, divided into types, NIH3T3 is better, 3T3L1 is more difficult to turn.

For difficult-to-transfect cells, I saw a cost-effective RFECT small nucleic acid transfection reagent on the Internet, which uses nanomaterials to make siRNA easier to transfer into cells, and has a high transfection positive rate for difficult-to-transfect cells. I have tried several cell lines that are difficult to transfect, and the effect is okay, and there is no problem with ordinary cell transfection.

With the addition of pyruvate, growth is accelerated.

It's also good if you don't lengthen it.

Most people are currently using lipofectamin 2000!! The transfection effect of this reagent is relatively good, especially when the virus is packaged, three plasmids are needed, so the requirements for the transfection reagent are higher.

Content from user: xieexin

Cell Transfection Experiment 1 Purpose of the experiment.

Learn and master the main method of introducing exogenous genes into eukaryotic cells—liposome-mediated transfection. Understand the general method of foreign gene entry, observe the expression of foreign protein (green fluorescent protein), and prepare experimental materials for staining.

2 Experimental Principles.

transfect

Shown above is a schematic diagram of lipid-mediated transfection, which shows the general process of exogenous plasmid entry into cells. There are four main methods for foreign genes to enter cells: electric shock, calcium phosphate, liposome-mediated and virus-mediated.

The electroshock method is a short-term temporary perforation of the cell to allow the entry of an exogenous plasmid; The calcium phosphate method and liposome method use different carrier substances to carry plasmids to make foreign genes enter the cell through direct membrane penetration or membrane fusion; The virus method is a method of infecting cells with viruses that package foreign genes so that they can enter the cells. However, due to the strict control of the experimental conditions of the electric shock method and the calcium phosphate method, it is more difficult. The preliminary preparation of the virus method is more complicated

And it may have a large impact on cells; Therefore, for many common cell lines, the general transient transfection method is mostly lipid method. Therefore, the ratio of liposomes to plasmids, cell density, the length of transfection time and the content of serum in the medium are all important issues affecting the transfection efficiency.

The figure above is a schematic diagram of the structure of the cationic components in the liposomes used in this experiment. The liposomes used in this experiment were Promega's TransFast Liposome Reagent, a mixture of cationic liposomes and neutral liposomes, which was optimized for transfection of 293T cells used in this experiment.

3.Experimental materials and equipment: 1) Materials: MYOD expression plasmid and EGFP expression plasmid D in 293T cells

The presence of serum can interfere with the mixing of siRNA with transfection reagent, thus affecting transfection efficiency. The recommended medium for diluting siRNA and diluting transfection reagents is serum-free medium. In the front section of our laboratory, we just finished using rfect and siRNA method to make A549 cells, the plating density of the cells was about 45%, and the final concentration of siRNA was set at 10 nm, 30 nm, 50 nm, and 100 nm in a 24-well plate, 2 double wells were made for each gradient to facilitate comparison.

This is a problem with the transfection reagent, which is slightly more toxic and causes cell death, so instead of serum-free medium, directly use complete medium without bispecific antibodies, or try rfect srna transfection reagent, which can not change the medium after transfection, and is less toxic to cells.

If 293T does not express this protein itself, the most likely is that the quality of the primary antibody is not up to par.

You can do a western blotting to see. Immunohistochemical false-positives are mostly normal (non-specific adsorption).

Western blotting is judged by molecular weight, which is difficult to fake.

The transfection toxicity of liposomes is the greatest, and it is clear that so much cell death is due to the cytotoxicity of the transfection. I have two suggestions for this, one is to reduce the amount of DNA, I usually do transfection, for any cell, as long as it is a 12-well plate, I usually use 1ug of DNA, this amount is definitely enough. Because the nature of plasmid transfection into the cell, no different from viral infection, the various elements of the plasmid will snatch a large number of cell machines from the host cell to start transcribing and translating your target protein, and in the process will produce a large number of proteins that are not folded properly, and these abnormal folded proteins will further cause er stress and lead to apoptosis.

So if you use too much DNA, it's inherently cytotoxic. The other is the toxicity of liposomes themselves. In fact, for the 293 line of the cell line, the best thing to use is something like pei.

The toxicity is low, the transfection efficiency is high, and it can easily reach 80% for the 293 series cell line. Polyscience has a powder to buy, and after buying it, directly prepare it into a 1ug ul solution in water of molecular purity, and transfect your plasmid with 1ug DNA 6ug pei for 293.

And the direct use of homemade pei is very cheap, far better on 293 than commercial liposomes.

In addition, if your lab really has a lot of money and is not afraid of spending money, it is recommended that you take Promega's Fugenhd, which has better transfection efficiency than liposomes, and is less toxic. Also higher ...

In addition, you say that floating cells have a surface to GFP, which is not necessarily true GFP, and many times.

Transfect DNA? 293t no matter how it turns, it gets better... Whichever is fine...

x 10 5 cells per well.

This is the advice given in the LIPO2000 manual.

If you use other transfection reagents, the instructions should have the recommended amounts, but they won't vary much.