Background[1-3]
The Calcium Phosphate Cell Transfection Kit is an improvement on the traditional calcium phosphate cell transfection method, improving transfection efficiency and reducing toxicity. Thermoplastic elastomers are transfected into cells using the calcium phosphate method. Not only can they be expressed transiently, but stable strains can also be screened.
HEK293 or HEK293T cells, commonly known as 293 cells, are one of the most suitable cells for calcium phosphate transfection. After optimizing conditions, the transfection efficiency can be as high as over 90%; it is usually easy to reach about 40-50%. Transfection efficiency.
Most common cells such as HeLa cells, CHO cells, etc. are also suitable for calcium phosphate transfection, but the efficiency is slightly lower than that of 293 cells. The Calcium Phosphate Cell Transfection Kit is not only suitable for the transfection of most adherent cells, but also for the transfection of some suspension cells. The reagents provided in the Calcium Phosphate Cell Transfection Kit are sterile and can be used directly.
Operation steps
1 Passaging Cell Preparation Cells are passaged 24 hours before transfection, and transfection can be performed when the cell density reaches 50% to 60%. 3 to 4 hours before adding the precipitate, culture the cells with 9 ml of complete culture medium.
2 Preparation of DNA precipitation solution: First, precipitate plasmid DNA with ethanol (10-50 μg/10cm plate), dry the precipitate in the air, resuspend the DNA precipitate in μL sterile water, and add 50 μL 2.5 mol/L CaCl2.
3 Use a Pasteur pipette to add the DNA-CaCl2 solution dropwise to 500 μL 2×HeBS, and at the same time pipe the solution with another pipette until the DNA-CaCl2 solution is dripped. The whole process needs to be carried out slowly and lasts for at least 1~ 2min.
4 Leave it at room temperature for 30 minutes, and fine particles will precipitate.
5 Add the precipitate drop by drop evenly into the 10cm plate and shake gently.
6 Culture cells under standard growth conditions for 4 to 16 hours. Remove the culture medium, wash the cells twice with 5 ml of 1×HeBS, and add 10 ml of complete culture medium to culture the cells. Collect cells or place into culture dishes for selective culture.
Apply[4][5]
Research on the induction and optimization of induction system for human iPS cells
A series of optimizations have been carried out on the induction system of human iPS cells, providing a solution that is simple to operate, does not require complicated equipment, and is not expensive. The specific research content and results are as follows: Calcium phosphate method and QuickShuttle transfection reagent were used to Viral vectors (FU-tet-o-hSox2, FU-tet-o-hOct4, FU-tet-o-hc-Myc, FU-tet-o-hKLF4, FUdeltaGW-rtTA) and retroviral vector pEYK E4 were used for viral The virus titer was packaged and measured, and the effects of different packaging systems, different transfection times, and the presence or absence of gelatin coating on the virus prepared using the calcium phosphate method and the QuickShuttle transfection reagent were studied.
Design orthogonal experiments from the above three aspects to explore optimal transfection conditions. The results show that whether the virus is prepared by the calcium phosphate method or the QuickShuttle transfection reagent, the 3-plasmid packaging system of pLP1, pLP2 and pLP/VSVG is used, and the transfected 293T cells are plated on a gelatin-coated culture dish. , and then transfected for 72 hours to obtain ideal results. Among them, the virus titer obtained by the calcium phosphate transfection method is 600TU/mL, and the virus titer obtained by the QuickShuttle reagent transfection method can reach 7130000TU/mL.
hFFCs and MEFs were primary cultured using single cell culture method and tissue block culture method. The results showed that the cell morphology of the primary cells obtained through tissue block culture method was better, and there were very few suspended dead cells after passage. . After MEFs were treated with mitomycin C, feeder cells were successfully prepared. hFFCs were infected with virus and their morphological changes were observed.
References
[1]New perspectives on the biology of fragile
[2]Modeling Parkinson’s Disease Using Induced Pluripotent Stem Cells[J].Blake Byers,Hsiao-lu Lee,Renee Reijo Pera.Current Neurology and Neuroscience Reports.2012(3)
[3]LRRK2 Mutant iPSC-Derived DA Neurons Demonstrate Increased Susceptibility to Oxidative Stress[J].Ha Nam Nguyen,Blake Byers,Branden Cord,Aleksandr Shcheglovitov,James Byrne,Prachi Gujar,Kehkooi Kee,Birgitt Schüle,Ricardo E .Dolmetsch,William Langston,Theo D.Palmer,Renee Reijo Pera.Cell Stem Cell.2011(3)
[4]Highly Efficient miRNA-Mediated Reprogramming of Mouse and Human Somatic Cells to Pluripotency[J].Frederick Anokye-Danso,Chinmay M.Trivedi,Denise Juhr,Mudit Gupta,Zheng Cui,Ying Tian,Yuzhen Zhang,Wenli Yang,Peter J.Gruber,Jonathan A.Epstein,Edward E.Morrisey.Cell Stem Cell.2011(4)
[5] Cheng Teng. Induction of human iPS cells and optimization of induction system [D]. Inner Mongolia University of Science and Technology, 2014.
