Gene Expression in Mammalian Cells Using BacMam, a Modified Baculovirus System

Fornwald, James A.; Lu, Quinn; Wang, Dayuan; Ames, Robert S.

doi:10.1007/978-1-59745-457-5_5

Cited by 35 publications

(19 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The use of polyspecific EcTyrRS and MbPylRS facilitates the incorporation of several useful UAAs using a single suppressor virus, including UAAs with bioorthogonal handles for chemical conjugation, photoaffinity labels, and others. Baculovirus, especially the pseudotyped version, has been shown to efficiently transduce a wide range of mammalian cells both in vivo and in vitro, all of which now should be suitable hosts for UAA mutagenesis of target proteins (9)(10)(11)(12)(13)(14)(15). The unique baculovirus vectors developed in this study should also be useful for other applications, where multiple genetic elements need to be simultaneously delivered into mammalian cells.…”

Section: Resultsmentioning

confidence: 98%

“…AcNPV is also able to infect some mammalian cells, where its genetic elements remain silent rendering it replication incompetent. Thus, it can be safely used to deliver genetic cargo to a variety of different mammalian cell types both in vitro and in vivo (9)(10)(11)(12)(13)(14)(15). Several properties of baculovirus make it attractive as a potential delivery vector for the UAA incorporation machinery, including its very large cargo capacity (>30 kb), stable double-stranded DNA genome, broad host-tropism, ease of production, long shelf-life of the purified virus, intrinsically safe nature, and minimal cytotoxicity to mammalian cells, even when high multiplicity of infection (MOI) is used (9)(10)(11)(12)(13)(14)(15).…”

Section: Resultsmentioning

confidence: 99%

“…A UAAs incorporated using OMeYRS (1-9) and MbPylRS (10)(11)(12)(13)(14) B Incorporation of 2 in GFP* in various cells Incorporation of pAcF 2 in eGFP (Tyr39TAG) using pAcBac1.tR4-OMeYRS baculovirus in various cells. Cells were infected with the suppressor and the reporter virus at an optimal 1:6 ratio (MOI of 800) and were analyzed 48 h postinfection.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

Chatterjee

Xiao

Bollong

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

109

132

View full text Add to dashboard Cite

Here we report the development of a baculovirus-based delivery system that enables the efficient incorporation of unnatural amino acids into proteins in mammalian cells. We have exploited the large cargo-capacity (>30 kb) and stability of the double-stranded DNA genome of baculovirus to deliver to a variety of cell types all of the components required to genetically incorporate novel amino acids. These include the engineered tRNA/aminoacyl-tRNA synthetase pair and the nonsense mutant of the target gene. Mammalian cell transduction efficiency of baculovirus was significantly improved by incorporating genetic elements from mammalian viruses. Two polyspecific tRNA/aminoacyl-tRNA synthetase pairs were inserted into this expression system, enabling the site-specific incorporation of a variety of unnatural amino acids with novel chemical and biological properties into proteins.genetic code | viral vector | nonstandard amino acid | orthogonal

show abstract

Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

Chatterjee

Xiao

Bollong

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

109

132

View full text Add to dashboard Cite

show abstract

“…The Smo BacMam virus was constructed and amplified based on published methods (Fornwald et al, 2007). The coding region for the Smo gene, cloned in-house from human ovary, aligns with National Center for Biotechnology Information sequence NM_005631.3.…”

mentioning

confidence: 99%

Evidence for Allosteric Interactions of Antagonist Binding to the Smoothened Receptor

Rominger¹,

Bee²,

Copeland³

et al. 2009

J Pharmacol Exp Ther

Self Cite

View full text Add to dashboard Cite

show abstract

“…The commercial use of the baculovirus (BV) expression vector system in the manufacture of human vaccines [1,2] and the exploitation of BV as benign non-replicative vectors for gene expression in mammalian cells [3][4][5] has generated a renewed interest in the technology. This technology is not without its challenges.…”

Section: Introductionmentioning

confidence: 99%

Development and validation of a HPLC method for the quantification of baculovirus particles

Transfiguracion

Mena

Aucoin

et al. 2011

Journal of Chromatography B

View full text Add to dashboard Cite

A HPLC method using an anion exchange column was developed for the quantification of baculovirus particles. To properly detect the virus eluting from the column, a nucleic acid dye was used to amplify the signal projected by the virus. The viral genome was labeled by incubating the virus with SYBR Green I at 37 • C for a minimum of 1 h. The virus was specifically eluted from the contaminants in 8.9 min at a NaCl concentration of 480 mM NaCl (in 20 mM Tris-HCl, pH 7.5). The total run time of the method was 25 min. The method resulted in a linear response from 1 × 10 8 to 5.0 × 10 10 viral particles (VP/ml). The detection limit was 3.0 × 10 7 and the quantification limit was 1 × 10 8 VP/ml. The intra-assay precision was <10% for both purified and crude virus preparations whereas the inter-assay precisions were <5% and <10% for purified and crude virus preparations, respectively. The recovery/accuracy of the method ranged from 78 to 101%. This method is a robust monitoring tool to facilitate research activities with baculovirus vector and accelerate development of baculovirus-based processes for manufacturing of biologics.Crown

show abstract

Gene Expression in Mammalian Cells Using BacMam, a Modified Baculovirus System

Cited by 35 publications

References 25 publications

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

Evidence for Allosteric Interactions of Antagonist Binding to the Smoothened Receptor

Development and validation of a HPLC method for the quantification of baculovirus particles

Contact Info

Product

Resources

About