DNA显微注射法 z-bo 2020-10-14 345

　　DNA microinjection is the earliest, most mature and most widely used transgenic technology. Other methods are improvements and supplements to this method. With the help of a micro-manipulation system, the foreign gene is directly injected into the fertilized egg of the donor animal, so the foreign DNA is integrated into the genome to produce a transgenic animal. The following mainly introduces the pronucleus microinjection method of fertilized egg. The basic process of DNA microinjection method includes preparation of transgenic vector, embryo manipulation and microinjection, and identification and cultivation of transgenic animals.

　　Micromanipulation requires key equipment, such as inverted microscopes, micromanipulation systems, microinjection systems, stereoscopes, needle forging instruments and microsurgery instruments. The inverted microscope must be equipped with DIC function. Commonly used brands are Zeiss and Nikon. The advantage of Differential Interference Contrast Microscopy (DIC) is that it can display three-dimensional structures and help inject DNA directly into the nucleus. The micromanipulation system mainly includes 3D electrical coarse micromanipulator, 3D hydraulic micromanipulator, needle holder, needle holder holder and microscope holder. The main brands are Eppendorf and Narisige. The Eppendorff microscope operating system consists of TransferManNK2, CellTramvario (for transfer and infusion), CellTramAir (for fixing fertilized eggs) or CellTramOil (for fixing embryos). The microinjection device uses FemtoJet/FemtotipⅡ, which can remove a small amount (about 1 to 2 pl). The DNA, RNA and protein of the fertilized egg are injected into the male cell nucleus. The auxiliary equipment of the microinjection system includes laser rupture system or piezoelectric mechanical perforation, which is suitable for thinning shingles in embryos of various species, and can significantly improve the success rate of ES cell transfusion. I can. Operate under a stereoscope to obtain and rinse embryos.

　　1. Preparation of the transgenic vector After constructing the transgenic vector, the vector must be digested with restriction enzymes, and the transgenic fragments must be separated by agarose gel electrophoresis at an appropriate concentration. The DNA gel recovery kit is used to recover DNA fragments from the gel and measure the DNA concentration. The injected DNA concentration is usually very low, usually 2-3g/μl.

　　2. Embryo manipulation and microinjection DNA microinjection usually requires injection of a large amount of oocytes containing hormones (pregnant horse serum PMSG and human chorionic gonadotropin, hCG) to stimulate ovulation. Have. At a specific time point, 4-6 weeks old donor mice (FVB/DBAF1 or C57BL/6) were injected with hormones (PMSG/hCG). One day before the microinjection, donor female mice and male mice were mated to obtain fertilized eggs, and embryo recipient mice were also required. One day before the DNA microinjection, C57BL/6 female mice were mated with ligated male mice. The next morning, the mice were examined, and the female mice with vaginal plugs were pseudo-pregnant mice. On the day of injection, the over-ovulating female mice were dissected, the ovaries and Faropius tubes were cut open, and fertilized eggs were collected. Digest the perlite with hyaluronidase and place the egg cells in a drop of embryo medium. Adjust the micromanipulator and needle to move the DNA solution to the micromanipulator. The basic principle of microinjection is to use a glass pipette with a smooth surface to suck out the fertilized egg from the microscope, and use another very thin glass needle to suck the DNA solution into the male pronucleus of the fertilized egg. It is direct injection. The injected fertilized eggs are transferred to the oviduct of the recipient animal and grow into offspring. The advantages of microinjection are simple, fast and fast, which is now the classic method.

　　The needle is so thin that it limits the size of the DNA you inject. The longer the DNA fragment, the more difficult it is to prepare transgenic animals. The most commonly used is plasmid DNA, which is approximately 10 kb in size. DNA over 50 kb is difficult to microinject, and DNA over 400-500 kb is difficult to microinject to obtain a complete transgene.

　　BACDNA microinjection: BAC is a circular DNA that can be separated with DNA extraction kits (such as QIAGEN). Resuspending and protecting the BAC DNA sample with an appropriate buffer increases the possibility of complete transfer of the BAC sequence to the genome. PFGE is used to test the molecular integrity of purified BAC DNA. Microinjection is usually used to inject BACDNA directly into the pronucleus of a fertilized egg. The effective concentration range of BACDNA for microinjection is very small, usually 0.5g/μl. If necessary, the concentration can be reduced to increase the survival rate of fertilized eggs and increase the birth rate. Due to the large size of BAC DNA, the shear force generated during injection will damage the DNA. It is necessary to identify the resulting transgenic mice by PCR and Southern Brotting to determine whether they contain the complete BAC gene.

　　The injected DNA is usually integrated into a single site in the cluster, and the number of copies ranges from a few to hundreds. After inserting multiple copies of DNA, it can be used to screen transgenic animals with high expression of foreign genes. Normal transgenic animals use plasmids as vectors, but the disadvantage is that the ability to clone plasmid DNA is too small to clone expression control elements, such as large genes and locus control elements. These transgenic animals should be called "hemidigas" rather than "heterozygotes" because they do not have corresponding allogenes on the homologous chromosomes corresponding to the integration site. The number of transgene integrations, that is, the number of transgene copies, is usually inversely proportional to the size of the DNA fragment. Therefore, the larger the DNA fragment, the more difficult it is to successfully integrate. Due to the random integration of transgenes in prokaryotic injection, transgene expression is usually affected by genes surrounding the insertion site. Due to different integration sites, each transgenic animal exhibits different levels of transgene expression. These effects can lead to gene silencing, change the specific expression of transgenes in cells and tissues or affect the overall expression level. When the transgene is incorporated into the euchromatin region, the surrounding endogenous enhancer enhances the transgene expression. When a transgene is incorporated into a heterochromatin region, heterochromatin-mediated silencing occurs and the expression level is reduced. .. Studies have shown that integration sites can subtly affect the expression of transgenes in the brain. When a transgene is inserted into an endogenous gene site, it can change the expression of the endogenous gene at the integration site or inactivate the insertion. In fact, it is possible to infer the role of the insertion site by analyzing multiple adult mice. The effect of the insertion site can also be reduced by adding an insulator sequence to the transgenic fragment.