Expression Of Proenkephalin Gene In NIH3T3 Cells

In patients with chronic pain, pain cannot be relieved by a number of conventional analgesics used for the treatment of chronic pain, therefore, the possibility of using ENK as potential novel analgesics is taken into consideration. In animal models of chronic pain, treatments with ENK had been succeed.Upon transfection into a cell line , the retroviru vector can integrate the proenkephalin gene into the cell line and stably express . pLNCX2 does not contain the structural genes (gag , pol , and env)necessary for particle formation and replication;and the viral cannot replicate within these cells since the cells lack the viral structural genes . The separate introduction and integration of the stractural genes into the packaging cell line minimizes the chances of producing replication-competent virus due to recombination events during cell proliferation. This virus produced by the stable virus packaging cell line transfect cell line NIH3T3, the cell line NIH3T3 can integrate the proenkephalin gene and stably express. Objective1. To find out if proenkephalin gene can acquired by RT-PCR.2. To find out if viral clones with high titers were picked out.3. To find out if the proenkephalin gene can express stably.Methods1. RT-PCRThe primers were designed based on the rat proenkephalin cDNA sequence in Genebank.The incoporration of /findlH and Clal restriction endonuclease site into 5' and 3' Primer allow the insertion of the PCR Products into retroviral vector. Total RNA was isolated. First strand of pENK cDNA synthesis reactions were performed using cDNA kit according to the manufacturer's recommendation. PCR amplification of pENK cDNA.2. The sequence read on the autoradrographThe PCR product was purified from agarose gel. Ligation with T-vector . The recombinant clones were selected by color screening on IPTG/X-gal Plates, and the white clones were picked out grown for preparation of cDNA. After the digestion with HindlH and Clal, the recombinant plasmid was indentified.Nucleotide sequencing of cloned fragments was done by the Sanger' s dideoxy-mediated termination using T7 sequencing kid. The sequence of the target DNA from the pattern of bands was read on the autoradrograph.3. To construction the retrovirus vector pLNCX2-ENKThe PCR product was digestion with Nindlll and Cla I then the cDNA fragment was inserted into plasmids. The constructed vector was transformed into E. coli JM109 competent cells, and were selected by the digestion with Nindlll and Cla I for inserts of the appropriate size.4. Packaging and selectingTransfomation of pLNCX2 into packaging cell line PT67 with lipofectamine 2000, performed using Invitrogen Transfomation kit according to the manufacturers recommendation, for 48 hour. Toselect the stable virus producer lines, cotransfected cells were transferred in selective medium containing 400mg/l of G418. After ten days cultivating, the G418 resistant clones were picked out and select eight clones for determining their viral titers. 5. Determining the viral titersMouse fibroblasts cell line NIH3T3 were cultured in complete medium. The virus was added in the medium, and incubated 2 days before selection. Virus infected cells were placed in the selective medium containing 400 mg/L G418 at limited dilution, incubated for an additional 4 to 7 days until colonies were visible, calculate the clones.6. The neorgene was cloned by PCR, to find out if proenkephalin gene can integrate into the NIH3T3 cell line. The concentration of enkephalin(ENK)in cell culture medium was measured with radioimmunoassay, the mRNA of preproenkephalin gene was also examined by RT-PCR. Results1. Rat proenkephalin gene was cloned by RT-PCR . The PCR product was purified from agarose gel. Ligation with T-vector. the recombinant plasmid was indentified. The sequence of the target DNA from the pattern of bands was read on the autoradrograph.2. The PCR product was digestion with Hind\\\ and Cla I , then the cDNA fragment was inserted into plasmids. The recombinant retrovirus vector pLNCX2-Enk was constructed.3. A stable virus producing packaging cell line with high CFU (8X105CFU/ml) was established.4. The proenkephalin gene can integrate into cell line NIH3T3 and stably express established by radioimmunoassay andRT-PCR.. Conclusions1. Rat proenkephalin gene can acquired by RT-PCR.2. Infection of NIH3T3 cell lines with retrovirus, The higher viral titers clones were acquired.3. The proenkephalin gene can integrate into cell line NIH3T3 and stably express.