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

• Published in: Electronic Journal of Biotechnology Vol. 22; no. 4; pp. 75 - 80
• Main Authors: Zhang, Jinju, Zhang, Yun, Liu, Xiaomei, Xiang, Jingjing, Zhang, Chun
• Format: Journal Article
• Language: English; Portuguese
• Published: Elsevier España, S.L.U 01.07.2016; Pontificia Universidad Católica de Valparaíso; Elsevier
• Subjects: BIOTECHNOLOGY & APPLIED MICROBIOLOGY; Glial cell line-derived neurotrophic factor; Multiplicity of infection; Recombinant adeno-associated virus 2; Site-specific integration; Transduction; Site-specific integration; Glial cell line-derived neurotrophic factor; Transduction; Recombinant adeno-associated virus 2; Multiplicity of infection
• ISSN: 0717-3458; 0717-3458
• DOI: 10.1016/j.ejbt.2016.05.001

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). 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 1×104, 1×105, and 1×106 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 72h of transduction, the rate of EGFP positive cell was 22%, 45% and 49% at the MOIs of 1×104, 1×105 and 1×106, 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 180d of cultivation. A HEK293T cell line able to site-specifically integrate and stably express GDNF was established.