Mesenchymal stem cell for expressing related gene of neurotrophin family and application thereof
A technology of neurotrophic factors and mesenchymal stem cells, which is applied in the field of mesenchymal stem cells and their preparation, can solve the problems of impossibility and difficulty of gene therapy
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Embodiment 1
[0064] Example 1: Isolation and culture of bone marrow MSCs and transfection of human bone marrow MSCs with green fluorescent protein gene
[0065] The green fluorescent protein (GFP) gene is the only new reporter gene found so far that can be expressed in cells without the participation of other exogenous substrates. In this study, human bone marrow stem cells were transfected with CMV-GFP gene, and the expression of GFP in human bone marrow stem cells was observed.
[0066] 1. Materials
[0067] 1. Equipment: ultra-clean bench, horizontal centrifuge, inverted microscope and ordinary microscope. Centrifuge tubes and balances, bone marrow puncture needles and blood collection bags. Flow cytometer, drying box, gloves and surgical gown. Hydrometer (1.000-1.100, 1.100-1.200)
[0068] 2. Reagents: Meglumine Diatrizoate (Sigma Company), Hydroxymethyl Cellulose (Sigma Company), Cesium Chloride (Sigma Company), Polysucrose (Ficoll-400, Phamacia Company), Potassium Dihydrogen Phos...
Embodiment 2
[0095] Example 2: Cloning of human GDNF and BDNF genes and construction of their N-terminals
[0096] 1. Cloning of human GDNF gene and its N-terminal construction:
[0097] Using human brain tissue information ribonucleotide (mRNA) as a template, use Poly(T) primers under the action of reverse transcriptase to synthesize complementary deoxyribonucleotide (cDNA) of human brain tissue, and then use human brain tissue cDNA As a template, use an artificially synthesized forward primer containing the nucleotides encoding the human interleukin-2 secretion leader polypeptide and the N-terminal sequence nucleotides of the human mature GDNF gene and the C-terminal sequence nucleotides of the human GDNF gene The reverse primer was used for PCR amplification, and the human GDNF gene clone and its N-terminal reconstruction were carried out, and the endonuclease NheI and NdeI sites were introduced at its N-terminus, and the endonuclease BamHI and HindIII sites were introduced at its C-ter...
Embodiment 3
[0120] Example 3, Construction of co-expression GDNF and BDNF expression cassette pAAV-MCS vector
[0121] Co-expression of GDNF and BDNF expression cassette pAAV-MCS vector construction method ( Figure 9 , 10) comprises the following steps:
[0122] 1) Using the multiple cloning site of the pAAV-MCS vector in the AAV helper-free expression system, the GDNF gene was purified with endonuclease BamHI and Xbal1 nucleotide fragments, digested with BamHI and Xbal1, and the purified pAAV- After the ligation reaction of the MCS vector, the competent bacteria were transformed, amplified, spread on a petri dish containing appropriate antibiotics, and grown overnight in a 37°C incubator;
[0123] 2) Screen multiple clonal colonies in culture medium containing appropriate antibiotics, culture overnight at 37°C with shaking, extract and purify pAAV-MCS plasmid DNA the next day, and digest with BamHI and Xbal1 enzymes to determine the presence of BamHI and Xbal1. Plasmid DNA and clone s...
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