Ana Puglia

Carlos Pereira

Daniella Ventini-Monteiro

Nayara Santos

Renato Astray

BMC Proceedings

PubMed

Ana Lia Puglia

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Influence of recombinant S2 cell population enrichment on rabies virus glycoprotein expression and especific RNA and DNA quantities

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POSTER PRESENTATION Open Access
Influence of recombinant S2 cell population
enrichment on rabies virus glycoprotein
expression and especific RNA and DNA quantities
Nayara Santos*, Carlos Pereira, Daniella Ventini-Monteiro, Ana Lia Puglia, Renato Astray
From 5th Congress of the Brazilian Biotechnology Society (SBBIOTEC)
Florianópolis, Brazil. 10-14 November 2013
Background
Different strategies have been evaluated for increasing the
productivity of Drosophila melonogaster (S2) cells expres-
sing recombinant rabies virus glycoprotein (RVGP). The
maintenance of selective pressure during all the cultivation
time and new methods for population enrichment with
high level expressing cells are two methods which can
increase the productivity through increased expression of
RVGP gene. To understand how these methods can
improve glycoprotein expression, analysis of the quantities
of heterologous DNA and RNA were performed [1,2].
Methods
From S2MtRVGPHy cell population (untreated control)
three populations were generated: S2MtRVGPHy+Hy was
obtained after selective pressure using hygromycin for
2 weeks; S2MtRVGPHy-M2 and S2MtRVGPHy-M3 were
obtained after immunomagnetic enrichment of RVGP
expressing cells (MACS, Miltenyl Biotec), using rabbit
polyclonal antibody and mouse monoclonal antibody,
respectively [3]. Cell populations were induced with
CuSO4 for RVGP expression and submitted to cyclohexi-
mide (CHX) treatment before sampling in different
periods. Samples were analyzed by ELISA, qPCR and
qRT-PCR.
Results
All cell populations presented no significative differences
in RVGPDNA content as analysed by qPCR. Before
treating the cultures with CHX, cell populations showed
very similar amounts of RVGPmRNA and were expres-
sing RVGP at concentrations of 23.1, 31.9, 65.3 and 45.7
ng / 106 cells for S2MtRVGPHy, S2MtRVGPHy-M2,
S2MtRVGPHy-M3 and S2MtRVGPHy+Hy, respectively,
showing that both population enrichment strategies
were successful on improving RVGP expression. When
CHX was added to cultures, the amounts of RVGP
decreased probably due to degradation and translation
interruption [4]. As expected, RVGPmRNA levels
increased.
Conclusion
Immunomagnetic enrichment of RVGP expressing cells
and hygromycin treatment showed to be efficient on
increasing RVGP productivity. These methods are easy to
perform and may be used for additional selection of
recombinant S2 cell populations that are unable to clonal
selection because of absence of growth in low cell concen-
trations. As these methods not statistically changed the
amount of RVGPDNA copies / cell between the cell popu-
lations in study, the differences in RVGP expression could
be attributed to different transcription and translation
rates. For studying expression profiles, an inhibitor of
translation in eukaryotes (CHX) was added to cultures for
translation blockage. The amounts of accumulated
RVGPmRNA showed that cell populations exhibited dif-
ferent profiles of transcription and translation for glyco-
protein expression. While S2MtRVGPHy-M3 produced
the highest level of RVGP (65.3 ng/106 cells), it showed
the smallest level of RVGPmRNA accumulation (R = 5.7)
among all populations. As S2MtRVGPHy+Hy produced
the second highest level of RVGP (45.7 ng/106 cells), and
showed the highest RVGPmRNA accumulation level
(R = 499.4), probably cells present different rates of
processing due mainly to metabolic differences. Future
experiments undergoing more broadly kinetic evaluations
of RVGP and RVGPmRNA may contribute do better
understand these results.
Instituto Butantan, São Paulo, SP, Brazil
Santos et al. BMC Proceedings 2014, 8(Suppl 4):P230
http://www.biomedcentral.com/1753-6561/8/S4/P230
© 2014 Santos et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Acknowledgements
Fundação para o Desenvolvimento Administrativo (FUNDAP) and Instituto
Butantan.
Published: 1 October 2014
References
1. Astray RM: Expressão da glicoproteína recombinante do vírus rábico em
sistemas Drosophila melanogaster (S2) e Semliki Forest Virus (SFV) = L´
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Drosophila melanogaster (S2) et Semliki Forest Virus (SFV). Tese em co-
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Biotecnologia USP/Instituto Butantan/IPT, para obtenção do Título de Doutor
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2. Lemos MAN: Construção e Transfecção de vetores plasmidiais contendo
o gene da Glicoproteína do Vírus da Raiva (GPV) em células de
Drosophila melanogaster. Dissertação apresentada ao Programa de Pós-
Graduação Interunidades em Biotecnologia USP/Instituto Butantan/IPT, para
obtenção do Título de Mestre em Biotecnologia 2009, 70-72.
3. Miltenyi Biotec GmbH: MACS Separators user manual. MACS Miltenyi Biotec
2011, 2.2.1.2-2.2.1.4.
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Wanke V, Anrather D, Ammerer G, Riezman H, Broach JR, Virgilio CDe,
Hall MN, Loewith R: Sch9 Is a Major Target of TORC1 in Saccharomyces
cerevisiae. Molecular Cell 2007, 26:663-674.
doi:10.1186/1753-6561-8-S4-P230
Cite this article as: Santos et al.: Influence of recombinant S2 cell
population enrichment on rabies virus glycoprotein expression and
especific RNA and DNA quantities. BMC Proceedings 2014 8(Suppl 4):P230.
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Santos et al. BMC Proceedings 2014, 8(Suppl 4):P230
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Influence of recombinant S2 cell population enrichment on rabies virus glycoprotein expression and especific RNA and DNA quantities

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