ShRNA lentivirus for inhibiting expression of ALKBH1 as well as preparation and application of shRNA lentivirus
A technology of lentivirus and expression vector, which is applied in the field of biotechnology and gene therapy, can solve the problems of unclear expression regulation mechanism and stress cognitive impairment, so as to resist and treat stress cognitive impairment , increasing BDNF levels, maintaining neuronal survival and neurogenesis
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Embodiment 1A
[0031] Embodiment 1 ALKBH1-shRNA lentiviral vector construction and identification
[0032] Obtained the transcript sequence of ALKBH1 (NM_001102565.1) from NCBI, designed shRNA interference sites targeting ALKBH1, screened out 3 sites with higher scores, and chemically synthesized ALKBH1-shRNA oligo and negative control shRNA oligo (Shanghai Jikai Biological Co., Ltd. company), annealed to form a double strand, cloned into a lentiviral expression vector, and obtained a recombinant plasmid.
[0033] (1) Design of the interference target of ALKBH1
[0034] Obtained the transcript sequence of ALKBH1 (NM_001102565.1) from NCBI, designed shRNA interference sites targeting ALKBH1, and screened out 3 sites with higher scores. The target sequence is as follows:
[0035] PSC-1: 5'-gcCATCTGCATGACCCGAATA-3' (SEQ ID NO.1)
[0036] PSC-2: 5'-gaAATACTCAGCAGATCATTA-3' (SEQ ID NO.2)
[0037] PSC-3: 5'-cgAAGGCTATCCTGGATTTAT-3' (SEQ ID NO.3)
[0038] (2) Construction of ALKBH1-shRNA lentiv...
Embodiment 2
[0078] Example 2 ALKBH1-shRNA plasmid interference effect detection
[0079] Transfect the identified positive recombinant plasmid into HT22 cells, collect the cells to extract RNA and reverse transcribe to obtain cDNA, detect the effect of recombinant plasmid on the expression of ALKBH1 by real-time PCR experiment, select the plasmid with the most significant interference effect for further lentiviral packaging .
[0080] (1) Cell transfection
[0081] (1) Take HT22 cells in the logarithmic growth phase, inoculate them into 6-well culture plates at a density of 50%, and place them in 5% CO 2 , Cultivate in a 37°C incubator until the cell density reaches about 80%.
[0082] (2) According to the instruction manual of jetPEI transfection reagent (polyplus company), the mixture of plasmid and transfection reagent was prepared and added to the cells dropwise.
[0083] (3) Observe the state of the cells after 24 hours, and replace with fresh complete medium. After 48 hours, the...
Embodiment 3
[0110] Embodiment 3 lentiviral packaging and quality detection
[0111] 293T cells were co-transfected with ALKBH1-shRNA interference plasmid and VSVG and psPAX2 plasmids, and the cell supernatant was collected after 48 hours of culture, and the lentivirus was concentrated and purified by ultracentrifugation, and finally the virus titer was measured by fluorescence method.
[0112] (1) Culture of 293T cells
[0113] 1. Recovery of 293T cells
[0114] (1) Prepare DMEM medium (called complete medium) containing 10% FBS for the cultivation of 293T cells.
[0115] (2) Add 3 mL of complete medium into a 10 mL glass centrifuge tube.
[0116] (3) Take the cells out of the liquid nitrogen tank or -80°C refrigerator, quickly put them into a 37°C water bath, and shake them gently for 1-2 minutes to completely melt them.
[0117] (4) Take the cryopreservation tube to the ultra-clean table, and wipe the surface with alcohol cotton ball for disinfection. Add the cell suspension to the ce...
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