Development of a Baculovirus-based Packaging System for Efficient Recombinant Retrovirus Production
Session Number
Project ID: BIO 06
Advisor(s)
Dr. Tong-Chuan He, University of Chicago
Discipline
Biology
Start Date
17-4-2024 9:40 AM
End Date
17-4-2024 9:55 AM
Abstract
Retroviral vectors are commonly used for generating stable cells to express transgenes. However, packaging high-titer retroviruses is technically challenging due to variations in co-transfecting the packaging cells with multiple plasmids that express genes essential for retrovirus production, leading to inefficient and inconsistent virus production and dramatic virus titer fluctuations. The objective of this study is to investigate whether or not baculovirus (BV)-mediated delivery of gag-pol-env and VSV-G would produce high-titer retroviruses. If successful, such a system would significantly simplify retrovirus-making, which is crucial for effective stable transgene expression in biomedical research. Experimentally, the gag-pol-env and VSV-G expression cassettes were first cloned into BV transfer vectors, the resultant constructs verified by DNA sequencing. The BV transfer vectors were transformed into DH10Bac bacterial cells, which harbor the BV genome that expresses Tn7 transposases. After the Tn7 transposition reaction, positive (white) clones were selected from X-gal/IPTG blue-white agar plates. The BV vectors for gag- pol-env (pBV-gag-pol-env) and VSV-G (pBV-VSV-G) were obtained. After verification, these BV vectors were transfected into Sf9 insect cells to produce BV viral particles. We have successfully obtained BV-gag-pol-env and BV-VSV- G baculoviruses, and are working on amplification to create high titer BV viruses for function tests and retrovirus production.
Development of a Baculovirus-based Packaging System for Efficient Recombinant Retrovirus Production
Retroviral vectors are commonly used for generating stable cells to express transgenes. However, packaging high-titer retroviruses is technically challenging due to variations in co-transfecting the packaging cells with multiple plasmids that express genes essential for retrovirus production, leading to inefficient and inconsistent virus production and dramatic virus titer fluctuations. The objective of this study is to investigate whether or not baculovirus (BV)-mediated delivery of gag-pol-env and VSV-G would produce high-titer retroviruses. If successful, such a system would significantly simplify retrovirus-making, which is crucial for effective stable transgene expression in biomedical research. Experimentally, the gag-pol-env and VSV-G expression cassettes were first cloned into BV transfer vectors, the resultant constructs verified by DNA sequencing. The BV transfer vectors were transformed into DH10Bac bacterial cells, which harbor the BV genome that expresses Tn7 transposases. After the Tn7 transposition reaction, positive (white) clones were selected from X-gal/IPTG blue-white agar plates. The BV vectors for gag- pol-env (pBV-gag-pol-env) and VSV-G (pBV-VSV-G) were obtained. After verification, these BV vectors were transfected into Sf9 insect cells to produce BV viral particles. We have successfully obtained BV-gag-pol-env and BV-VSV- G baculoviruses, and are working on amplification to create high titer BV viruses for function tests and retrovirus production.