Studies of a TMV-based expression vector in plant cell cultures.

다국어 초록 (Multilingual Abstract)

Plant virus expression vectors for transient heterologous protein production have been developed and successfully used in whole plant host expression systems. However, there is currently no published work concerning the development of plant virus expression vectors in plant cell culture. We established and studied cell cultures infected with a TMV-based expression vector, 30Bcycle3GFP, containing the gene for Cycle3GFP under the control of a strong promoter. The infected cultures were derived from a variety of methods. First, we initiated infected cell cultures from plants infected with 30Bcycle3GFP. Second, 30Bcycle3GFP infected cell cultures were obtained using a direct inoculation technique. Third, infected cell cultures showing antibiotic resistance were developed by co-culturing infected cell cultures with transgenic cell cultures constitutively expressing a gene product for antibiotic resistance. Additionally, we examined the possibility of developing infected cell cultures from high-efficiency protoplast transfections with 30Bcycle3GFP. The production of Cycle3GFP, levels of viral vector RNA, and the insertional stability of the Cycle3GFP gene were characterized in the infected cultures.
We obtained fluorescing Cycle3GFP levels as high as 2.4% of the total soluble proteins (%TSP) extracted from infected cultures initiated from infected plants. In directly inoculated cell cultures, we achieved Cycle3GFP levels as high as 0.7% TSP extracted although infection efficiencies were low. Maximum Cycle3GFP expression levels of 4.9% TSP extracted were obtained in infected cultures exhibiting antibiotic resistance. Derivatives of the 30Bcycle3GFP vector that had lost the Cycle3GFP gene were found in several infected cultures and we provide evidence that some vector derivatives were replicating. There were no detectable levels of 30Bcycle3GFP vectors showing loss of insert in directly inoculated cultures examined one month post-inoculation. In addition, some infected cultures maintained Cycle3GFP fluorescence for generations with manual selection of fluorescing tissues during subculturings. Our data suggest that a selection process for viral vector infected cells would increase the stability of heterologous protein expression and reduce the heterogeneity of viral vector infection in our system. Furthermore, the ability to directly inoculate plant cell cultures with a viral vector shows the potential for a rapid, high-level heterologous protein expression system in plant cell culture.