Gene therapy refers to the suppression, replacement or supplementation of defective genes at the genetic level through the regulation of the expression of target genes, thereby restoring the physiological functions of cells, tissues and organs. There are two ways of gene therapy: in vivo (hWW) and ex vivo (ex v/vo). The in vivo approach is to introduce the target gene directly into the sacrificial cells in vivo to achieve therapeutic effect, while the ex vivo approach is to take out the cells of a certain organ or tissue from the patient's body, expand them in vitro, transfer the target gene into them, and build up the genetically modified cells expressing the exogenous genes, and then implant the cells back into the body with a certain number of cells to achieve the therapeutic effect after expansion and cultivation. The current gene therapy strategy is more in vitro approach. The selection of appropriate carrier cells to carry the therapeutic genes to the therapeutic location and to function is an important factor for the success of the treatment. Because stem cells not only have the ability of self-replication and renewal, which is conducive to the long-term stable expression of genes, but also are easy to take and easy to culture in vitro and implant back into the patient's body, in recent years, stem cells have been studied more as the target cells of gene therapy, and some of these therapeutic options have already entered the stage of clinical trials. Author: Xuedao Pei, this experiment is from "Stem Cell Experiment Guide".
Operation method
Stem Cell and Gene Therapy Move Experimental techniques for virus-mediated gene transduction Retrovirus-mediated gene transduction At present, in gene therapy, it is more common to see various types of retroviral carriers modified by Moloney mouse leukemia virus (MoLV), which retains the cis-functional structure, while knocking out the g, three-part pathogenic trans-functional sequence in the DNA gene structure and replacing the empty region with an exogenous gene; therefore, they are deficiency viruses, which lack the structural gene region of retroviruses, and are not able to code structural protein. Therefore, it is a defective virus, which lacks the structural gene region of retrovirus and cannot code the structural protein. Packaging cells are formed by transfection of mouse fibroblasts NIH3T3 with different helper viruses, which can provide structural proteins for retroviral vectors, and thus the two of them can compensate for each other and combine to package virus particles into progeny. Since this viral particle is still recombinant retroviral vector DNA, this daughter viral particle has different genetic material from that of the wild-type retroviral particle, so it is called a pseudoviral particle, and buds out of the membrane of the packaging cell into its culture supernatant. The protein component of the retroviral envelope contains a glycoprotein expressed by the M V gene, and many mammalian cells have specific receptors that recognize this glycoprotein; the combination of the two mediates the entry of the retrovirus into the host cell and its efficient integration into the host . . in the host cell chromosome D N A . Content of the experiment Packaging of recombinant retroviral vectors and clone screening 1 ) Culture of PA:317, N IH 3 T 3 cells After the cells have grown to their full size, pass them on as necessary. Passaging method: Remove the medium with steps, wash once with PBS liquid, add trypsin digest (0.5 ml for 50 ml culture flasks, and lml for IOOml culture flasks), shake the flasks gently, and let them stand for 1-3 min at room temperature or 37 ℃, then add an appropriate amount of serum medium to terminate the reaction, and then blow the flasks with the dropper repeatedly to make the cells fall into cell suspension. Blow the dropper repeatedly to dislodge the cells into cell suspension, inoculate the cell suspension into new culture flasks according to the required density, and incubate the cells in an incubator at 37℃, 5% C02. 2) Determination of the minimum lethal dose of G 148 on P.A. 317 cells. A 24-well plate was inoculated with the appropriate amount of P.A. 317 cells. When cells have grown to 50% to 60% confluence, selective culture medium containing different concentrations of G 418 (0_2 g/L, 0.3 g/L, 0.4 g/L, 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L) was added. Change the solution every 2 to 3 days, and determine the minimum lethal dose of G 418 to PA 3117 according to the cell death situation in a week or so. Insertion of target gene into adenoviral shuttle plasmid. The target gene (Y F G ) was inserted into the polyclonal site of adenoviral shuttle plasmid (PAdtrack-C M V ) by genetic manipulation, and then the recombinant was transformed into E. coli D H 5a, and the positive recombinant was subjected to P C R and enzyme digestion. The specific experimental steps were omitted. Screening and positive identification of monoclonal recombinant adenovirus (1) Inoculate 2 9 3 cells in a 6-well plate and proceed to the next step when the cells reach 80% fusion. (2) Dilute the supernatant of recombinant virus at 10 times ratio in Iml serum-free DME, aspirate the supernatant of 293 cells, add the virus dilution and incubate at 37 ℃ for lh. Materials and reagents Cell culture B H K cells were cultured in 1640 medium + 10 % calf serum + l ○ 0 U/m l penicillin + l ○ 0 m g /m l streptomycin. Note: Do not move the cells after the medium is removed, otherwise the pH will change and the transfection efficiency will be low. Calcium phosphate transfection Virus Concentration Gradient dilution of virus solution was added into 293T cells, and the expression of G FP was observed under a fluorescence microscope after 72 h. The number of incandescent cells was counted, and the titer was calculated according to the amount of virus and the degree of dilution. Lentiviral vector in vitro infection 1) H E K 293 2) AAV Vector Packaging System 3) AAV Helper Free Packaging System 4) OptiprepTM Isolation Solution (A XIS -S H IELD) For more product details, please visit Aladdin Scientific website.
The exogenous genes in retroviral vectors include target genes and marker genes. Marker genes are generally neomycin resistance genes, tachykinin genes, and puromycin genes, etc., but most of them are used, so G 418 can be used to screen and clone the transfected packaging cells.
Resuscitation method: After removing the cells from liquid nitrogen, shake them vigorously in a water bath at 371℃ for 1 min, aspirate the cell suspension into a centrifuge tube, then dropwise add 8~IOml of medium, centrifuge at 10000 r/min for 5 min, discard the supernatant, and then dilute the cells with appropriate medium (DMEM medium containing 10% fetal bovine serum, 2mol/L glutamine, l0 U/m I penicillin, and lOOpg/m l streptomycin). After dilution, the cells were inoculated into culture flasks and cultured, and the medium was changed to fresh medium on the following day, and then every 2~3 days after that, and the cells were passaged as needed after full growth.
Adenovirus-mediated gene transfer 
Materials and reagents 
Content of the experiment 
Virus titer measurement 
Chronic virus-mediated gene transfer
293T cells were cultured in high sugar DM EM medium, and the preparation of culture medium was as follows: DMEM+10% bovine serum+100U/ml penicillin+100U/ml streptomycin. After 24 h of cell propagation, remove 2 ml (100 Cm flask) or 5 ml (15 cm flask) of medium and leave the cells in the incubator for at least one hour.
Lentiviral titer assay
1) FUGW titer assay
2) The titer of FUXW and FAXW was determined by Southern spot hybridization.
H E K 293 is an adherent-dependent epithelial cell containing human embryonic kidney cells of the A d 5 E l region, grown in D M E M medium (containing 10 % FBS). It is a packaging cell for adeno-associated virus.
These include p A A V - M S C (containing an exogenous gene rhyming polyclonal site for insertion of the target gene), helper plasmids (e.g., A d 8), adenoviral helper genes or helper viruses (e.g., A d 5), and packaging cells (e.g., H E K 2 9 3 ).
Includes pAV-MSC, pHelp Plasmid and packaging cells (e.g., HEX 23). The p Helper Plasmid is supplied directly with
complementary and helper genes. It contains the essential adenoviral genes V A E 4 E 2 A and the packaging genes for r A A V (which, cap), so that no adenovirus is required to provide the helper genes. To package r V V virus particles, the recombinant A V V vector can simply be cotransfected with the plasmid into E l-containing 23 cells, thus avoiding the contamination of the wild-type A d .
Iodixanol, a highly hydrophilic, nonionic substance, is the main component that makes OptiprepTM Isolates a dense, low viscosity solution. Moreover, its density and permeability form a linear relationship. After adding appropriate concentration of buffer or basic medium, such as sucrose, HEPES or PMI, OptiprepTM can form a continuous or discontinuous isotonic density gradient solution, which can separate various kinds of cells, organelles and lipoproteins. It has been proved by scientific research and clinical experiments that OptiprepTM is not only better than conventional separating solutions such as Ficoll, Percoll, Sucrose and CSCL, but also has no effect on the life activity of cells and organelles. The technical parameters of the product are Iodixanol: 60% (m /V ); Density :y(1.320±0.001)g/ml; Osmolality: (170±15)mOsmo. 
