[Construction of recombinant adenovirus vector of calcitonin gene-related peptide gene and transfection to neonatal rat cardiomyocytes]

Zhi-hui Sun, Jie Han, Lei Shao, Li-hong Wang, Jun-xian Song, Zhong-hai Wei, Liang-rong Zheng
Zhejiang da Xue Xue Bao. Yi Xue Ban, Journal of Zhejiang University. Medical Sciences 2010, 39 (3): 311-7

OBJECTIVE: To construct a recombinant adenovirus vector of calcitonin gene-related peptide (CGRP) by AdEasy system and to validate its expression in myocardial cells.

METHODS: The full-length of CGRP gene cDNA was acquired by RT-PCR and cloned into pShuttle-CMV. After linearization with Pme I, the recombinant plasmid (pShuttle-CMV-CGRP) was transformed into E.coli BJ5183 by electroporation to construct the recombinant adenovirus plasmid AdEasy-pShuttle-CGRP. The recombinant adenovirus plasmids were transformed into E.coli XL10-Gold cells to be amplified. Then the recombinant plasmid was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. PCR technique was used to detect target gene. The recombinant adenovirus particles were purified by CsC1 density gradient. The purified recombinant adenovirus was transfected to neonatal rat cardiomyocytes,and the recombinant adenovirus production was observed by fluorescent microscope. Expression of CGRP in hearts 7 days after intravenous delivery of adenoviral vectors AV-CGRP was determined by radioimmunoassay.

RESULT: The RT-PCR products confirmed a full-length cDNA of CGRP gene in PUC(57) by sequencing. The corresponding double endonuclease and PCR analysis certified the successful cloning of the gene into the pShuttle-CMV. The recombinant adenovirus plasmid AdEasy-pShuttle-CGRP was digested by Pac I endonuclease to form the typical DNA segments, whose length was about 3 kb and 30 kb. PCR analysis and fluorescent microscope observation confirmed that the CGRP gene was inserted into the adenovirus vector with very strong power of transfection. The recombinant adenovirus particles infected neonatal rat cardiomyocytes successfully. Radioimmunoassay showed that delivery of AV-CGRP significantly increased the expression of CGRP in mice hearts.

CONCLUSION: The recombinant adenovirus vector of CGRP gene has been constructed,and it can infect neonatal rat cardiomyocytes successfully. Somatic delivery of CGRP gene can significantly increase the expression of CGRP in mice hearts. The results may provide a sound foundation for further study on the value of CGRP as the target for gene therapy in both laboratory and clinical trials.

Full Text Links

Find Full Text Links for this Article


You are not logged in. Sign Up or Log In to join the discussion.

Related Papers

Remove bar
Read by QxMD icon Read

Save your favorite articles in one place with a free QxMD account.


Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"