Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector

Background: Using recombinant adeno-associated virus 2 (rAAV-2), we attempted to establish a HEK293T cell line that is able to site-specifically integrate and stably express glial cell line-derived neurotrophic factor (GDNF). Results:Recombinant vector with enhanced green fluorescent protein (EGFP)...

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Autores principales:	Zhang,Jinju, Zhang,Yun, Liu,Xiaomei, Xiang,Jingjing, Zhang,Chun
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2016
Materias:	Glial cell line-derived neurotrophic factor Multiplicity of infection Recombinant adeno-associated virus 2 Site-specific integration Transduction
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000400011
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spelling	oai:scielo:S0717-345820160004000112016-09-13Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vectorZhang,JinjuZhang,YunLiu,XiaomeiXiang,JingjingZhang,Chun Glial cell line-derived neurotrophic factor Multiplicity of infection Recombinant adeno-associated virus 2 Site-specific integration Transduction Background: Using recombinant adeno-associated virus 2 (rAAV-2), we attempted to establish a HEK293T cell line that is able to site-specifically integrate and stably express glial cell line-derived neurotrophic factor (GDNF). Results:Recombinant vector with enhanced green fluorescent protein (EGFP) and GDNF (pTR-P5-EGFP-IRES-GDNF), as well as that carrying Rep genes and SV40 promoters (pSVAV2) were constructed and packed. HEK293T cells were co-infected with rAAV-2/EGFP-GDNF and rAAV-2/SVAV2 virus separately at 1 x 10(4),1 x 10(5),and 1x10(6) of multiplicity of infection (MOI). The efficiency of transduction was detected using flow cytometry. Additionally, the infected HEK293T cells were separately validated by touchdown polymerase chain reaction (PCR) and Western-blot. After 72 h of transduction, the rate of EGFP positive cell was 22%, 45% and 49% at the MOIs of 1 x 10(4),1 x 10(5) and 1 x 10(6), respectively. On the 3rd, 6th and 9th day of cell passage, there was no significant difference in the cell viability and proliferation rate between transduction and control groups. Importantly, touchdown PCR showed that there was a specific PCR amplified product band in the lane of infected cells. Furthermore, GDNF expression was detected in the infected cells after 15 and 180 d of cultivation. Conclusions: A HEK293T cell line able to site-specifically integrate and stably express GDNF was established.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.19 n.4 20162016-07-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000400011en10.1016/j.ejbt.2016.05.001
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	Glial cell line-derived neurotrophic factor Multiplicity of infection Recombinant adeno-associated virus 2 Site-specific integration Transduction
spellingShingle	Glial cell line-derived neurotrophic factor Multiplicity of infection Recombinant adeno-associated virus 2 Site-specific integration Transduction Zhang,Jinju Zhang,Yun Liu,Xiaomei Xiang,Jingjing Zhang,Chun Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector
description	Background: Using recombinant adeno-associated virus 2 (rAAV-2), we attempted to establish a HEK293T cell line that is able to site-specifically integrate and stably express glial cell line-derived neurotrophic factor (GDNF). Results:Recombinant vector with enhanced green fluorescent protein (EGFP) and GDNF (pTR-P5-EGFP-IRES-GDNF), as well as that carrying Rep genes and SV40 promoters (pSVAV2) were constructed and packed. HEK293T cells were co-infected with rAAV-2/EGFP-GDNF and rAAV-2/SVAV2 virus separately at 1 x 10(4),1 x 10(5),and 1x10(6) of multiplicity of infection (MOI). The efficiency of transduction was detected using flow cytometry. Additionally, the infected HEK293T cells were separately validated by touchdown polymerase chain reaction (PCR) and Western-blot. After 72 h of transduction, the rate of EGFP positive cell was 22%, 45% and 49% at the MOIs of 1 x 10(4),1 x 10(5) and 1 x 10(6), respectively. On the 3rd, 6th and 9th day of cell passage, there was no significant difference in the cell viability and proliferation rate between transduction and control groups. Importantly, touchdown PCR showed that there was a specific PCR amplified product band in the lane of infected cells. Furthermore, GDNF expression was detected in the infected cells after 15 and 180 d of cultivation. Conclusions: A HEK293T cell line able to site-specifically integrate and stably express GDNF was established.
author	Zhang,Jinju Zhang,Yun Liu,Xiaomei Xiang,Jingjing Zhang,Chun
author_facet	Zhang,Jinju Zhang,Yun Liu,Xiaomei Xiang,Jingjing Zhang,Chun
author_sort	Zhang,Jinju
title	Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector
title_short	Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector
title_full	Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector
title_fullStr	Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector
title_full_unstemmed	Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector
title_sort	establishment of a hek293t cell line able to site-specifically integrate and stably express gdnf by raav-2 vector
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2016
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000400011
work_keys_str_mv	AT zhangjinju establishmentofahek293tcelllineabletositespecificallyintegrateandstablyexpressgdnfbyraav2vector AT zhangyun establishmentofahek293tcelllineabletositespecificallyintegrateandstablyexpressgdnfbyraav2vector AT liuxiaomei establishmentofahek293tcelllineabletositespecificallyintegrateandstablyexpressgdnfbyraav2vector AT xiangjingjing establishmentofahek293tcelllineabletositespecificallyintegrateandstablyexpressgdnfbyraav2vector AT zhangchun establishmentofahek293tcelllineabletositespecificallyintegrateandstablyexpressgdnfbyraav2vector
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Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector

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