Method for differentiation of dopaminergic neuron by SHH/Lmx1a signal pathway regulation and control NTN modified rASCs
A dopaminergic and signaling pathway technology, applied in the field of biomedicine, can solve the problems of rising incidence of PD, failing to fundamentally restore the normal function of the lesion, and limiting the long-term treatment of Parkinson's disease
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[0049] 1. Cloning of Lmx1a gene and chimeric NTN gene and construction of recombinant plasmid
[0050] Using Lmx1a whole transcript cDNA (NM_138396) as a template, the primers are:
[0051] P1 up: 5'-GCGTCGACGTACCATGCTGGACGGCCTAAAG-3',
[0052] P1 down: 5'-CGGGATCCCGCTTATCAAGATGTGAAGTAAG-3',
[0053] The DNA fragments with SalI and EcoRV restriction sites were amplified by PCR, detected and analyzed by 1% agarose gel electrophoresis, and the amplified bands were consistent with the theoretical molecular weight. The results are shown in figure 1 a. The amplified fragment was inserted into pShuttle-CMV, and the recombinant clone was identified by double digestion with SalI and EcoRV. The results are shown in figure 1 b. The recombinant clones were sequenced, and the sequencing results were consistent with the theoretical results.
[0054] The above Lmx1a whole transcript cDNA (NM_138396) was produced by GeneCopoeiaTM.
[0055] Connect the NTN mature peptide gene to the art...
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