动物模型,基因修饰 z-bo 2020-08-14 502

　　1. Traditional ES targeting gene knockout: mature technology, accurate modification, stable effect, but the production cycle is as long as one year

　　The homologous recombination technology based on embryonic stem cells is commonly known as "traditional gene targeting technology". Because of its mature technology, accurate modification, stable effect and other advantages, it has been praised by many scientists. In addition, all important mouse animal models in the world are prepared by this technology. Although emerging nuclease knockout technologies such as ZFN, TALEN, CRISPR/Cas9, etc. have appeared one after another, homologous recombination technology based on embryonic stem cells is still the only technology that can meet all genome modification requirements.

　　Gene targeting is to integrate a foreign gene into a certain location on the target cell genome through homologous recombination technology to achieve the purpose of modifying a certain gene on the chromosome. Gene targeting technology has been widely regarded as an ideal method for specific modification and modification of the genetic material of organisms, including a variety of different gene knockout and knock-in systems, especially conditional and inducible gene targeting The establishment of the system makes the modification of the target position of genes in time and space clearer, and the effect is more accurate and reliable.

　　2, TALEN gene knockout: short cycle, but there is mosaic phenomenon and off-target phenomenon

　　TALEN technology is a new molecular biology tool. Scientists discovered that the amino acid sequence of the nucleic acid binding domain of the TAL protein from a plant bacteria has a constant correspondence with the nucleic acid sequence of its target site. Using the sequence module of TAL, it can be assembled into a modular protein that specifically binds to any DNA sequence, so as to achieve the purpose of targeted operation of endogenous genes. It overcomes the inability of the ZFN method to identify any target gene sequence, and the recognition sequence is often affected by upstream and downstream Sequence effects and other issues, and have the same or better flexibility of ZFN, making gene manipulation easier and more convenient. However, because of the off-target problem, genetic modification of mice using TALEN technology still cannot replace traditional techniques. In addition, there is mosaic phenomenon, so when doing gene knock-in (KI) and other applications, it must be carefully tested.

　　3. CRISPR/Cas9 gene knockout: short cycle, but mosaic phenomenon and off-target phenomenon

　　CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) is the latest technology in which RNA guides Cas nuclease to perform specific DNA modifications on targeted genes. CRISPR is an adaptive immune defense mechanism developed by bacteria and archaea to respond to the constant attacks of viruses and plasmids. In this system, crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA) through base pairing to form double-stranded RNA. This tracrRNA/crRNA binary complex directs the Cas9 protein to cut at the target site of the crRNA guide sequence. Cut double-stranded DNA to achieve the purpose of modifying genomic DNA. The CRISPR/Cas9 system can accurately edit specific gene sites in mouse and rat genomes. It has been successfully applied to the preparation of KO/KI models of rat and mouse genes. The principle is that after the target fragment is specifically cut, the repair process is in progress. Frameshift mutations or fragment deletion, or fragment insertion at the incision position.

　　4. TetraOneTM gene knockout: mature technology, accurate modification, stable effect, and the production cycle is only 6 months

　　TetraOneTM gene knockout is a new technology introduced by the industry. It follows the technical system of embryonic stem cell homologous recombination, and uses unique lineage and genetic modification technology to establish a genetically superior TetraOneTM ES cell line. Through microinjection in the early stage of embryonic development, Make TetraOneTM ES cells 100% replace the endogenous ES cells, realize the "chimera" stage, and quickly obtain the patented gene targeting technology of de-Neo heterozygous mice.

　　TetraOneTM technology is based on the gold standard of embryonic stem cell technology. Through homologous recombination technology, the foreign gene is integrated into a certain position in the target cell genome to achieve the purpose of modifying a certain gene in the genome. At the same time, TetraOneTM technology also has the advantage of the short cycle of nuclease technology, shortening the cycle of traditional ES targeting technology by half, and will not bring off-target effects and mosaic phenomena like nuclease technology. Before nuclease technology is mature, TetraOneTM technology will It is the best choice for researchers.