Method and device for assisting in gene transfer by utilizing electroporation
A technology of gene transfer and electroporation, which is applied in the field of ophthalmology, can solve the problems of complex cell hierarchy and difficulty in design and application, and achieve the effects of convenient operation, avoiding cell immunogenicity and potential pathogenicity, and low side effects
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Embodiment 1
[0046] Example 1 Preparation of double spoon type special electrode device
[0047] On the basis of the conventional ophthalmic electrode device, gold is used as the conductive contact surface material of the electrode, copper-aluminum alloy is used as the support material, and the outer layer is coated with resin as the insulating layer, and the specifications of the obtained electrode shape are guaranteed by measurement, double spoon type electrode( figure 1 ) Its two poles are connected to the electrical stimulation generator through a plug, and the positive and negative poles can be adjusted as required. The cornea-side electrode of the double-spoon electrode is in the shape of a large round spoon, which is mainly in line with the area and curvature of the cornea; the electrode on the scleral side is slightly smaller, in the shape of a small round spoon, and is only placed at the plasmid transfer site, which can reduce the separation range of the retrobulbar area and Red...
Embodiment 2
[0049] 1. Construction of plasmid vectors
[0050] The existing plasmid pRc / CMV BDNF contains mouse BDNF cDNA, and the fragment length is 750bp. The primers of sequence 1 and sequence 2 (5'-ATG ACC ATC CTT TTC CTT ACT ATG-3' and 5'-CCA CTA TCT TCC CCT TTT AAT GG-3') were designed and synthesized. PCR reaction (94°C for 30s, 57°C for 30s, 72°C for 40s, 30 cycles) amplified the target gene, electrophoresed and recovered to obtain BDNF cDNA. Then, according to the instructions provided by NT-GFP Fusion TOPO TA Expression Kits, the connection of BDNF cDNA and pcDNA3.1 / NT-GFP-TOPO vector plasmid, transformation, plasmid extraction, and sequencing were carried out. After sequencing confirmation, use the Endofree Plasmid Purification Maxi kit to extract the plasmids and detect the OD260 / OD280 value of the obtained plasmids. If the ratio is greater than 1.8, it is qualified, and the qualified plasmids are diluted to a concentration of 2.5 μg / μl.
[0051] 2. Microsurgery experiments ...
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