转基因 z-bo 2020-08-13 642

　　In the process of developing transgenic mice or gene-embedded reporter gene model mice, reporter genes are often used. Commonly used reporter genes include: lacZ, EGFP, zsGreen, EYFP, DsRed (DsRed2), mRFP, mCherry, HuCD2, Thy1.1, HA tag, FLAG tag, Luciferase, etc. Then when to choose which reporter?

　　Researchers working on mouse embryonic development generally like to use LacZ, which means that LacZ is placed under the regulation of a promoter. LacZ can be used for whole body staining of embryos, so that you can understand the full picture of the expression profile of a gene in the body. If you do cell tracking, such as immune cells, nerve cells, etc., you generally like to use fluorescent protein. One is that it is easy to separate cells (such as FACS) and in vitro analysis, and the other is that it is easy to track the separated cells in vivo (such as adoptive transfer). In the early years, EGFP and EYFP were used the most. Sometimes, we need to mate two different reporter mice together to study the relationship between the two genes. At this time, the two genes can be labeled with different fluorescent proteins, that is, to make two different model mice. . Since most people used EGFP in the early days, we can now make some other fluorescent protein reporter mice to combine with the previously developed EGFP mice. zsGreen and EGFP, EYFP are all green fluorescence. EGFP and EYFP are difficult to distinguish. zsGreen is a relatively stable green fluorescent protein. Both DsRed and TdTomato are red fluorescent. Both of these fluorescent proteins have been published in reporter gene model mice. TdTomato is a fluorescent protein with a very strong signal. It has been published in mice with reporter genes and has no obvious toxicity to cells/mouses. I believe that more and more mice will use TdTomato as reporter genes in the future. Although mRFP and mCherry are two very good fluorescent proteins, they require 584nm excitation light, so ordinary FACS instruments cannot detect the signal, and instruments such as LSRII are needed. Many laboratories may not have such instruments. EBFP and ECFP are also used for cell experiments, but they are rarely used on mice. For example, ultraviolet light is needed for excitation. If you want to express two fluorescent proteins in a mouse, it is best to use two separate ones. For example, it is best not to use EGFP and EYFP at the same time.

　　HuCD2 and Thy1.1 are cell surface receptors. HuCD2 has lost its signal transduction function because of the removal of the C-term sequence. When will these two molecules be used? For example, if we want to study the function of a protein gene (such as a transcription factor) in a cell, we often need to isolate and purify the cell expressing this protein. At this time, you can place the HuCD2 or Thy1.1 reporter gene under the promoter of the intracellular protein gene (for example, add an IRES-Thy1.1), so that all cells expressing this intracellular protein will express Thy1.1 on the cell surface. In this way, the anti-Thy1.1 antibody can be used to isolate this group of cells for subsequent research. Of course, anti-Thy1.1 antibody can also be used for immunofluorescence detection of tissue sections.

　　HA, FLAG, Biotin, etc. are commonly used tag peptides or proteins in cell and biochemical experiments. It is often used in western blot, immunoprecipitation and other experiments because of the very specific HA and Flag antibodies. Biotin can be directly combined with biotin (Streptavidin) and used for immunoprecipitation. In model mice, these tag peptides or proteins can be made into fusion proteins with the protein to be studied. This model of mice is most commonly used in Chip-chip experiments. For example, to study the function of a transcription factor, it is necessary to know the position and binding sequence of the transcription factor in the cell (under normal or stimulating conditions) that bind to the chromosomal genomic DNA. We know that antibodies against transcription factors with good specificity and high affinity are very difficult to obtain or prepare. At this time, the tag polypeptide (such as FLAG) can be placed in the C-term of the transcription factor to make a fusion protein. After the cells are stimulated, the cells are chemically cross-linked, and the cells and genomic DNA are disrupted, and then immunoprecipitation is performed with anti-FLAG antibody to precipitate the transcription factors. Finally, the DNA fragment sequence to which these transcription factors are combined is analyzed to find the recognition sequence of the transcription factor on the genome, and the gene of the transcription factor is also found.

　　Luciferase is used as a reporter gene model mouse, usually for in vivo imaging experiments. For example, place the luciferase gene under a cytokine promoter. After immunizing mice, some cells or organs express the cytokine and express luciferase. The distribution of fluorescence in mice can be detected by injection of luciferase substrate, and the expression of the cytokine under different immune conditions can be obtained. Luciferase is also frequently used in cancer research. Fluorescence in vivo imaging can study the occurrence and metastasis of tumor cells.

　　There are many reporter genes. Here is just a brief introduction. Different models of mice can have very different designs according to different experimental purposes.