[Expression of enhanced green fluorescent protein in transduced variant HT-29c cells in vitro and in vivo].
OBJECTIVE
To evaluate the gene transfer and expression of enhanced green fluorescent protein (EGFP) in retrovirally transducted variant HT-29c cells in vitro and in vivo.


METHODS
The retroviral vector prkat EGFP/neo was constructed and was transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (in vivo selected HT-29 cells) were transduced by a retroviral vector encoding the EGFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transfected with the EGFP gene bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransfected and transfected in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo.


RESULTS
After being transduced, HT-29c cells with the EGFP gene displayed a stable and long-term EGFP expression under the nonselective conditions in vitro. After culturing cells successively to passage 50 in vitro, EGFP expression level was still high. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransfected parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed.


CONCLUSIONS
An EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo. These cells are a valuable tool for in vivo analysis of metastatic spread.
Department of Surgical Oncology, First Affiliated Hospital, Medical College, Zhejiang University, Hangzhou 310003, China.
Min
M
Wang
Boenicke
B
Lars
Howard D
HD
Bradley
Vogel
V
Ilka
Kalthoff
K
Holger
D000818:Animals; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004195:Disease Models, Animal; D015870:Gene Expression; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D019073:HT29 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D009362:Neoplasm Metastasis; D009368:Neoplasm Transplantation; D051381:Rats; D011923:Rats, Nude; D014161:Transduction, Genetic; D014162:Transfection
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins