Lin Zhang1, Bao-hua Liu, Wei-dong Tong, Chun-xue Li. 1. Department of General Surgery, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China.
Abstract
OBJECTIVE: To investigate the construction of the green fluorescent protein (GFP) labeled recombinant adenovirus containing human stem cell leukemia (hSCL) and its distribution and efficiency in mice with interstitial cells of Cajal (ICC) loss. METHODS: The recombinant adenovirus Ad-GFP/SCL was constructed by Ad-Easy system based on the homologous recombination in bacteria, then 1.6 x 10(9) PFU of recombinant adenoviruses were injected into Balb/c mice with ICC loss via the tail vein. In vivo distribution and efficiency of recombinant adenoviruses mediated hSCL were observed by GFP under the fluorescent microscope at different phases. The expression of SCL gene was measured by RT-PCR method. The damages were observed in different organs by HE staining. RESULTS: The recombinant adenovirus containing hSCL was quickly constructed by homologous recombination in bacteria using Ad-Easy system. Under the fluorescent microscope, GFP was revealed in heart, lung, liver, spleen, kidney, small intestine and large intestine of mice with ICC loss at different phases. No obvious damages were observed in various visceral organs by HE staining. RT-PCR showed SCL mRNA expression in various visceral organs at different levels. CONCLUSIONS: Construction of adenovirus vector by the homologous recombination in bacteria is an efficient and time saving method, and a high titer of adenovirus is able to mediate the safe and stable expression of SCL gene in mice with ICC loss. This finding will make further gene therapy in mice with STC possible.
OBJECTIVE: To investigate the construction of the green fluorescent protein (GFP) labeled recombinant adenovirus containing human stem cell leukemia (hSCL) and its distribution and efficiency in mice with interstitial cells of Cajal (ICC) loss. METHODS: The recombinant adenovirus Ad-GFP/SCL was constructed by Ad-Easy system based on the homologous recombination in bacteria, then 1.6 x 10(9) PFU of recombinant adenoviruses were injected into Balb/c mice with ICC loss via the tail vein. In vivo distribution and efficiency of recombinant adenoviruses mediated hSCL were observed by GFP under the fluorescent microscope at different phases. The expression of SCL gene was measured by RT-PCR method. The damages were observed in different organs by HE staining. RESULTS: The recombinant adenovirus containing hSCL was quickly constructed by homologous recombination in bacteria using Ad-Easy system. Under the fluorescent microscope, GFP was revealed in heart, lung, liver, spleen, kidney, small intestine and large intestine of mice with ICC loss at different phases. No obvious damages were observed in various visceral organs by HE staining. RT-PCR showed SCL mRNA expression in various visceral organs at different levels. CONCLUSIONS: Construction of adenovirus vector by the homologous recombination in bacteria is an efficient and time saving method, and a high titer of adenovirus is able to mediate the safe and stable expression of SCL gene in mice with ICC loss. This finding will make further gene therapy in mice with STC possible.