Expression of the respective constructs has been tested in recombinant hosts in bacteria, insect cells, and mammalian cells. The host cells and their commercial sources are listed below:
Bacterial Expression Host Strains:
BL21(DE3)pLysS: Promega, cat. no. L1191
ArcticExpress (DE3): Agilent Technologies, cat. no. 203192
Tuner™(DE3)pLysS: EMD4Biosciences, cat. no. 70624
Baculovirus expression protocols, host cells, and media
Detailed protocols for baculovirus expression can be found HERE.
The vast majority of the expression assays on the baculovirus vectors produced in this project were performed using Sf9 cells as the host. Sf9 is a lepidopteran insect cell line clonally derived (Summers, M.D. and Smith, G.E. 1987. Texas Agricultural Experiment Station Bulletin #1555) from IPLB-SF21-AE ("Sf21"), a cell line originally isolated from the ovaries of Spodoptera frugiperda (fall armyworm) pupae (Vaughn et al., 1977. In Vitro 13:213). In addition, there are other lepidopteran insect cell lines that can be used as hosts for baculovirus vectors.
Representative sources of some of these cell lines include:
| Cell line | Vendor source |
| Sf9 | ATCC (CRL-1711) Invitrogen (Cat #B82501) EMD (Cat #71023 TriEx™ Sf9 cells) |
| Sf21 | Invitrogen (Cat# B82101) Clontech (Cat #631411) |
| "High Five™" (BTI Tn 5B1-4) cells (from Trichoplusia ni): | Invitrogen (Cat #B85502). |
Representative types and sources of insect cell growth media include:
| Media | Vendor Source |
| TNM-FH (aka Hink’s TNM-FH) | Sigma Aldrich BD Biosciences HyClone (Thermoscientific) |
| ESF921 (serum-free) | Expression Systems |
| Sf900 (serum free) and Express Five (serum free) | Invitrogen |
| EX-CELL (serum free) | Sigma Aldrich |
| SFX and SFM4 (serum free) media | HyClone (Thermoscientific) |
Technical guidance on insect cell culture can be obtained in any one of several excellent baculovirus expression system "manuals":
1. Summers, M.D. and Smith, G.E. 1987. A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station Bulletin #1555.
2. O'Reilly, D.R., Miller, L.K., and Luckow, V.A. 1992. Baculovirus Expression Vectors. W.H. Freeeman and Company, N.Y.
3. Murhammer, D.W. (ed.). 2007. Baculovirus and insect cell expression protocols. Second ed. Methods in Molecular Biology Vol 388. Humana Press, New Jersey.
Mammalian expression protocols, host cells, reagents
Detailed protocols for transient transfection of HEK293 cells can be found HERE.
Cell lines:
Freestyle™ 293-F cells from Life Technologies (Cat. No. R790-07)
HEK293S GnTI- cell line (ATCC cat. no. CRL-3022)
Media, transfection reagents, and additives:
Freestyle™ 293 Expression Medium from Life Technologies (Cat. No. 12338026)
Ex-Cell® 293 Serum-Free Medium (Sigma cat. no. 14571C)
ESF serum free medium (Expression Systems cat. no. 98-001)
Polyethylenimine (PEI) Polysciences, Inc. (Cat. No. 23966)
VPA (Sigma Cat. No. P4543)