A method for preparing human definitive endoderm cells
A technology for stereotyping endoderm and cells, which is applied in the field of stem cell induction and regenerative medicine, can solve the problems that the nature and differentiation characteristics of endoderm cells have not been studied, and achieve the effect of saving consumables, reducing costs, and high costs
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Embodiment 1
[0048] Example 1 Cloning of GDF3 gene into TetOne-Modified lentiviral vector
[0049] 1. TetOne-Modified plasmid construction:
[0050] TetOne-Modified plasmid is in the pLVX-TetOne-Puro plasmid (Clontech company, picture see figure 1 ) from the skeleton. A segment of the SV40 polyAsignal sequence on the pLVX-TetOne-Puro plasmid was removed, and the SV40 promoter that initiates the expression of puromycin was replaced with the T2A sequence. See the schematic diagram of the transformed pLVX-TetOne-Puro plasmid figure 1 .
[0051] The present invention finds that not all transformed plasmids are suitable for transformation into animal cells and achieve good results. After screening through a large number of experiments, the inventors found that the transformation of the above plasmids can be transformed into mammalian cells and achieved excellent results. Both the SV40 polyA signal sequence and the SV40 promoter sequence are derived from the SV40 virus, and if they are transf...
Embodiment 2
[0060] Example 2 Packaging of TetOne-Modified-GDF3 lentivirus
[0061] Kit used: Maichen Technology’s enhanced calcium phosphate transfection reagent, containing HBS and CaCl 2 .
[0062] 1. Human embryonic kidney epithelial cell line Commercial 293T cells can be purchased from ATCCA Company for passage: 8×10 6 293T cells were plated in a ten-centimeter cell culture dish, and the medium was high-glucose DMEM plus 10% fetal bovine serum (Fetal Bovine Serum, FBS, Vistech). Observe the cell shape, and the cells with good growth state have better encapsulation effect. The normal growth form of 293T cells is that the cells are plump, with about three or four protrusions, and there are few particles in the cells. The boundaries between cells are obvious, and there is no aggregation or fusiform shape. Virus packaging can be carried out when the cell confluency reaches 80--90%.
[0063] The following operations were performed in a biological safety cabinet (Esco).
[0064] 2. Pre...
Embodiment 3
[0070] Example 3 TetOne-Modified-GDF3 lentivirus infection of iPS cells
[0071] 1. Recovery, culture and passage of iPS cells
[0072] Geltrex coating: Geltrex (Thermo Fisher Scientific) was placed on ice to melt, and then diluted to 2 mg / ml with ice-cold DF12 (Thermo Fisher Scientific). Add 5ml to a 10cm petri dish and place at 37°C for 30 minutes to 4 hours.
[0073]Preparation of PGM1: PGM1 medium was purchased from Saibei Biology, and the supplement was melted at 4°C, and added to the basal medium to obtain PGM1 medium. Take 12ml of PGM1 and add 5 μM small molecule Y27632 (Selleck, CAS number: 129830-38-2). Heat the water bath to 37°C. Take out the frozen iPS cells, purchased from clonetech company, put them into the water bath quickly, shake gently until the ice cubes are completely melted. Dry the cryovials. Gently transfer the cells to a 15ml centrifuge tube. Add 5ml of PGM1 containing Y27632 dropwise, and shake gently while adding. Centrifuge at 1000 rpm for 3 ...
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