Report gene classification and advantages and disadvantages
A reporter gene is a gene encoding a protein or enzyme that can be detected, and its expression product is very easy to identify. The coding sequence and the gene regulatory sequence are fused and expressed under the control of the control sequence, thereby using its expression product to calibrate the expression regulation of the target gene. The conditions that must be met as a reporter gene: (1) The entire sequence has been determined (2) The expression product is absent in the hand-held cells, ie, has no background, and there is no similar endogenous expression product in the transfected cells. (3) Its expression product can be quantitatively determined There are many types of reporter genes, which can be divided into in vitro reporter genes and in vivo reporter genes depending on the analytical methods of their expression products. The former analysis requires the quantification of reporter molecules in cell lysates or cells containing tissue molecules, tissue culture fluids (secretory reporter genes). Typical examples include the chloramphenicol acetyltransferase gene (CAT), the human growth hormone gene (hGH), and the secreted alkaline phosphatase gene (SEAP). The latter analysis can be performed in living cells or tissues, or in tissues or cells that have been tissue-fixed. More typical such as the green fluorescent protein gene (GFP). The β-galactosidase gene (β-Gal) and the firefly luciferase gene can be analyzed both in vitro and in vivo. Commonly used reporter gene systems: √ galactosidase reporter system √ luciferase reporter system √Fluorescent Protein Reporting System: Green fluorescent protein (GFP) : Soluble protein, stable in nature; The fluorophore is quite bright; Fluorescence can be observed in living cells without the need for fixation and tissue processing; After the GFP group and the gene of interest are made into a fusion protein, they can be stably expressed in cells by transfection; Cells expressing the GFP fusion are able to perform experiments and assays in a living state. Reporter gene advantage Disadvantage Chloramphenicol acetyltransferase gene (CAT) No endogenous activity in mammals; can be detected by automated ELISA Linear range is narrow; sensitivity is relatively low; radioisotope is required --galactosidase gene (β-Gal) Protein stable; sensitive to biological or chemiluminescence; no need to use isotopes Endogenous activity in mammalian cells Human growth hormone gene (hGH) Is a secreted reporter protein; easy to operate Relatively low sensitivity; narrow linear range Secreted alkaline phosphatase gene (SEAP) Chemiluminescence detection is very sensitive; linear range is wide; cheap Endogenous expression in certain cells; interference with screened compounds Luciferase gene (Luciferase) Good sensitivity; wide linear range; no endogenous expression; easy to measure Protein has a short half-life; routine analysis has poor repeatability Green fluorescent protein gene (GFP) Autofluorescence (no substrate required); no endogenous expression; multiple mutants available Need post-translation processing; low sensitivity (no signal amplification) 21" Sanitary Sleeve For Embryo Transfer 21 Inches Sanitary Sleeve,Disposable Sanitary Chemise,Embryo Transfer Cover,Et Sheath Cover Jinan Mucho Commercial Inc. , https://www.muchovet.com