Baculovirus-Mediated Expression in Mammalian Cells.

1. Baculovirus-Mediated Expression in Mammalian Cells. 5 / 27 / 2003 Jong-Chan, AHN Interdisciplinary Graduate Program in Genetic Engineering (IGPGE)

2. Enhancement and Prolongation of Baculovirus-Mediated Expression in Mammalian Cells: Focuses on Strategic Infection and Feeding Yu-Chen Hu,* Chien-Tai Tsai, Yao-Jen Chang, and Jen-Huang Huang Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan 300 Biotechnol. Prog. 2003, 19, 373-379 • The baculovirus/insect cell system has been widely used for recombinant protein production. • 2. In this study, They intended to explore the possibility of utilizing a baculovirus/mammalian cell system. • 3. A recombinant baculovirus vector carrying EGFP under the control of CMV-IEpromoter was constructed. • 4. HeLa was found to yield the highest expression level.

3. How did they treat it?

4. Construction of the plasmid pBac-CMV-EGFP

5. The egfp gene along with the upstream CMV-IE promoter was amplified by PCR from pEGFP-C1 plasmid (Clontech) using the following primers: 5¢-CGCG AGATCTTAG TTA TTA ATA GTA ATC AAT TA-3¢ 5¢-AAGCTT TTA CTT GTA CAG CTC GTC CAT GCC G-3¢ (enzyme sites Bgl II and Hind III are underlined). Where?

6. CGCG AGATCT pEGFP-C1 Bgl II AA AAGCTT Hind III

7. The PCR product was inserted into the BamH I/Hind III sites of pFastBac DUAL (Invitrogen, Carlsbad, CA), which placed the expression cassette in multiple cloning site (MCS) I under the polyhedrin promoter. The resultant plasmid was designated pBac-CMV-EGFP as shown before. This arrangement placed both polyhedrin and CMV-IE promoter in tandem and upstream of the egfp gene and conferred the EGFP expression in both insect and mammalian cells. BamH I Bgl II 5'-A^G A T C T-3‘ 3'-T C T A G^A-5' 5'-G^G A T C C-3‘ 3'-C C T A G^G-5' The polh-CMV-IE-EGFP expression cassette was transferred from pBac-CMV-EGFP to a bacmid in DH10Bac E. coli by site-specific transposition. pFASTBAC™ DUAL allows for the cloning and simultaneous expression of two heterologous proteins.

8. RESULTS • Bright-field (upper panel) and fluorescence (lower panel) micrographs of four cell lines (HeLa, Cos7, BHK, and CHO) infected by Bac-CE (24 hpi). • The infection was conducted in 12 well plates at MOI = 200 when cells were at 1 x105 cells/well. • Variation of the percentage of GFP+ cells infected by Bac-CE with time. • The percentage was measured by comparison of fluorescing and nonfluorescing cells. The virus infection did not result in apparent cell death or growth arrest; hence, the cells continued to divide, albeit at a slightly slower rate (compared with uninfected cells, not shown). Moreover, the virus does not naturally replicate in the mammalian cells (15); thus, the cell division resulted in a decrease in the percentage of GFP+ cells after 24 hpi. NONETHELESS…

9. It was observed that a significant portion of the new daughter cells also emitted fluorescence after 72 h , which suggested that the baculovirus genomes were distributed to the new cells during the cell division cycle. Time course profiles of the total FI in four cell lines infected by Bac-CE. [Fluorescing intensity (FI) of individual daughter cells] HeLa cells yielded the highest overall EGFP expression that culminated at 96 hpi.

10. How can we use it better ? Is it possible to make a good mammalian expression system ? So …

11. Let’s make a … Baculo-Tet off & Tet on – Multiple Epitope tagging mammalian Expression Vector !!!

12. Modified Baculo Donor plasmid 의 제작 First, Amplify. (Use PCR) And then,

13. Second, Remove Polh prometer at pFastBac1. Third, Cloning TRE & CMV promoter region at this position.

14. Is it OK?

15. Fourth, Amplify Red BOX. Defective enhancer elements !

16. Defective enhancer elements