反转录病毒载体感染法 z-bo 2020-10-14 307

　　Prokaryotic injection and gene targeting of ES cells have been very successful in mice, but these methods have not been very successful in other species, which prompted scientists to find other alternative methods. Studies have shown that retroviruses, especially lentiviral vectors, can effectively introduce foreign DNA into egg cells. The basic structure of a retroviral vector: Both ends of a retroviral vector have long terminal repeats (LTR).

　　"LTR" contains promoter and enhancer sequences that can transcribe and express foreign genes. After the 5'LTR sequence of the retroviral vector, there is a viral packaging signal, the packaging protein gene is removed, and the remaining space can be used to clone foreign genes. The cloning ability of retroviral vectors is limited, and the inserted DNA fragment must be less than 10 kb. The packaging protein gene is cloned into an expression vector to prepare a packaging cell line. Retroviral expression vectors are used to transfect packaging cell lines or co-transfected with viral packaging vectors to prepare viral particles, and then the viral supernatant is used to infect embryos or ES cells. let me. Since shingles constitutes a physical barrier and cannot directly infect the conjugate, the supernatant of the recombinant virus can be injected into the space between shingles and the progenitor cell membrane, or shingles can be removed first. Then, it can infect connecting cells without shingles. You can also use a microscope. The operating system is injected and the virus supernatant is injected into the blastocyst cavity to infect early embryos. The embryo is transferred to the oviduct of the recipient animal and grows into a transgenic animal. After virus infection, the retroviral RNA genome is released into Sufeng cells. The viral RNA is reverse-transcribed into DNA under the action of reverse transcriptase, and is randomly integrated into the DNA under the action of the virus integrase and its terminal specific nucleic acid sequence. In the host. will. Cell genome. Murine leukemia retrovirus (MMLV) infection can be used to prepare transgenic animals at an early stage, and then enter the animal genome to undergo MMLV-mediated gene transfer, that is, gene expression silencing. Find. Recently, it has been found that lentivirus-mediated gene transfer is more stable in animals. Retroviral vector infection method The transgenic method is easy to operate, has a wide host range, is not affected by the embryonic development stage, does not have the continuous phenomenon of introducing genes, and integrates a single site and a single copy with high integration efficiency.

　　The disadvantage of this method is the need to produce retroviruses containing foreign genes. The insertion length of foreign genes is limited. Retroviral integration only occurs in dividing cells, while recombinant lentivirus can be used to infect non-dividing cells. Many transgenic animal models, including the transgenic rat model of Alzheimer's disease, have become fertilized mouse eggs infected with lentiviral vectors.