基因型鉴定 z-bo 2020-10-14 429

　　Genotype (genetic type) refers to the type of genes related to the traits contained in the DNA of an organism. The genotypes of transgenic animals are mainly identified by PCR and Southern blot analysis. PCR reaction is a traditional method. For transgenic animals, Southern hybridization may be required to check the copy number of the introduced gene.

　　Southern hybridization can also be used to determine whether the introduced gene has been rearranged or deleted. When genes are rearranged, the restriction sites change, resulting in different restriction patterns. Real-time PCR (RT-PCR) can quickly determine the expression of transgenic mRNA. The transgene is randomly inserted into the genome of the animal. If the transgenic animal is normal, there is usually no need to locate the transgenic integrant.

　　For identification, the integrated position and local chromosome structure can be directly observed by fluorescence in situ hybridization (FISH) under a microscope. This technology can also be used to detect chromosomal rearrangements at transgene insertion sites and local chromosomal deletions induced by Cre recombinase. The principle of PCR reaction genotyping is to first design a specific primer that can combine with the introduced foreign gene and amplify the DNA sequence that does not exist in the animal genome. The primer design is very important. Primers for genotyping transgenic animals are usually found in transgenes. Usually, a specific DNA sequence is selected as a primer to determine whether there is a transgene. In most cases, the insertion site of the transgene is unknown, and it is difficult to design primers to determine whether the transgene is homozygous or heterozygous. For gene knockout and gene knock-in animals, the location of gene modification is well known, so wild primers and mutant primers can be designed to determine whether the genotype is homozygous or heterozygous. Mutant primer pair: One primer is on the outside of the short arm of homologous recombination, and the other primer is on the inside of the introduced foreign gene (such as Neo gene). Wild primers are usually found in knocked-out DNA sequences. The size of mutant and wild PCR products should vary between 100-700 bp. This is sufficient for identification and identification by gel electrophoresis. The GC ratio of the primer is about 50% (40%-60%).

　　When analyzing genotypes, please pay attention to the setting of the control group. Positive control of PCR reaction: optional endogenous housekeeping genes, such as β-actin, to determine DNA quality and correct amplification reaction system. The PCR reaction of each DNA sample must be positive. Negative control: A control group that usually replaces DNA template with water to eliminate contamination and false positives. Experimental process: Extract the DNA of transgenic animals, amplify the target sequence through PCR reaction and agarose gel electrophoresis, and detect the presence and size of DNA fragments to determine the genotype. In order to isolate genomic DNA from living tissue, the biological anatomical tissue of a mouse can be a tail or ear tissue. The genotyping of transgenic mice can only be analyzed with a specific pair of primers, and the result is positive () or negative (-). Positive means that there is an imported gene, and negative means that there is no imported gene. The identification of knockout or knock-in mouse genotype usually requires the design of two pairs of primers. One pair of primers amplifies the wild form, and the other pair of primers amplifies the variant. There are three types of genotype identification results. The wild type is represented by and the variant type is represented by -. /- is knockout heterozygote, / is wild mouse, -/- is knockout homozygote.

　　Identification of gene expression level: transgenic animal model. Various methods (PCR, Western blot, flow cytometry, etc.) can be used to determine the level of transgene transcription or protein expression. In gene knockout or knock-in models, it is necessary to observe whether the target gene is knocked out and whether the gene is expressed. Transcription level analysis can be achieved by Northern blotting, ribonuclease (RNase) protection analysis and non-quantitative RT-PCR. If quantification is required, it can be determined by RT-PCR. This method is easy to use and has high quantification accuracy. It is a common method for gene expression analysis. In situ hybridization can also be used to determine the level of mRNA expression in tissues and cells. Finally, identification should include analysis of protein products and their expression levels. This is related to the phenotype displayed by any animal model. Most protein analysis methods require the use of specific antibodies against gene products. These techniques include Western blotting, enzyme-binding immunosorbent assay (EUSA), radioimmunoassay (RIA), and immunohistochemical staining.