1. What is transient expression and stable expression?
Warm up before class, learn the students, please go directly to question 2
Transient expression: Exogenous fragments cannot replicate together with cell division, resulting in a short expression time , and the expression level gradually decreases with time.
Stable expression: refers to the integration of exogenous fragments into the genome of the cell, and can be stably transmitted with cell division, and the expression level is at a stable level for a long time.
The stable strain, as the name implies, is of course a system of stable expression.
2. Does my experiment need to screen stable strains?
The way to achieve stable expression is not just a stable strain. When we use a lentivirus to infect cells, we can achieve stable expression. What is the principle?
* Lentivirus is an integrated retroviral vector. After infection, the foreign fragment carried will be directly integrated into the genomic DNA of the cell and can be stably passaged with cell division. Therefore, for most experiments, functional experiments such as cell cycle, apoptosis, proliferation, and even long-term experiments such as tumor formation in nude mice, as long as the cell infection efficiency is high (70% to 80% or more), a lentivirus is infected. It can achieve the purpose of long-term and efficient gene manipulation, meet the experimental requirements, and does not need to screen stable cell lines.
3, the cell infection efficiency is higher (70% ~ 80% or more), the minister can not do, how to do difficult to infect cells?
Congratulations, you have chosen the hard mode. Then summon a stable strain. So the question is, what method is chosen to integrate the foreign fragment into the cellular genome?
Plasmid (conventional transfection/transformation): This method mediated the integration probability is similar to the 500w lottery (< 10 -6 ), and the size of the integrated fragment is random, often not lost watermelon (target gene) That is, sesame (resistance gene) is lost, resulting in a high false positive / negative rate.
Lentivirus: The foreign fragment carried is directly integrated into the genomic DNA of the cell by the action of the viral recombinase, so the efficiency is several orders of magnitude higher than that of the plasmid. And the integration fragment is fixed, and the target gene and resistance gene can finally go hand in hand.
The following is a standard process for screening for resistance genes.
* The key to the drug screening method is the determination of the concentration and time of the drug. This is pre-tested according to the different needs of the cells, and it is also ensured that the drug does not adversely affect the state of the cells while screening stable strains.
➢ Commonly used resistance screening markers: neomycin, hygromycin, and puromycin. G418 is used instead of neomycin for selective screening.
Recommended concentration of antibiotics ( μg/ml )
There are pits here. Please note that most of the cell lines are group animals. They need the same kind of companion to grow normally. If they are unlucky, they will grow slowly or simply, and they will not get the monoclonal strain.
4, then the problem is coming, the same is a stable strain, a single? Mixed clone? Silly points are not clear ~ how to choose?
Monoclonal stable strain: Amplification derived from a single cell containing a stably integrated exogenous fragment.
A mixed stable strain: a clonal population composed of a mixture of a plurality of monoclonal stable strains, and different monoclonals have different integration sites for exogenous fragments.
Due to the different backgrounds of different cell genomes, and the cell phenotypes of monoclonal cells with different integration sites may be inconsistent, the mixed clones can remove the interference caused by the difference between cells, otherwise it needs to be stable to multiple monoclonals. The strains are compared to obtain more accurate experimental data.
In order to remove the difference between cells and get more accurate experimental results, we recommend that you use lentivirus infection, or build a mixed clone stable strain!
â— Infection efficiency of 70% to 80% or more can be directly tested and will not be questioned
â— High scores and low scores all use mixed clones, international recognition
â— Screening of monoclonal strains requires at least two cells to remove intercellular differences, and the workload is doubled.
For example, in the following 5 articles, it took 2 months to construct a stable strain, and then the cell function and animal experiments were done twice. Not only must the expression of the target genes of the two monoclonal strains be consistent, but the functional test results should be similar! What if it is inconsistent? If you change another one and repeat it, I really dug a big hole for myself!
5. What should I do if the stable strain cannot be constructed? Stable strain that can induce expression: TET-on lentivirus system
In the following two cases, the conventional stable strain construction is more difficult, but the method is always more difficult.
1) Overexpression: look at gene function. If it is to inhibit the proliferation of tumor cells or promote the apoptosis of tumor cells, it is difficult to construct a stable strain (the target gene will be apoptotic or not long after infecting the cells).
2) Interference: It is recommended to construct a stable strain that can induce expression. RNA interference knockdown is a gene that is endogenously used by cells. After knocking down, the inhibitory effect will be seen in a short time. However, after a long time, there will be up-regulated genes in the cell that will be inhibited by regulatory mechanisms, even more than conventional. The amount.
In both cases, it is recommended that the virus infect the cells directly for functional experiments or for tumor formation in nude mice. If you want to make a stable strain, you need to use the TET-on lentivirus system.
So what is the TET-on system? How to use it specifically?
See you next time!
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