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

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• 作者：Jinju Zhang¹ ; Yun Zhang¹ ; Xiaomei Liu ; Jingjing Xiang ; Chun Zhang ; ^{chunzhdr@163.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Glial cell line-derived neurotrophic factor ; Multiplicity of infection ; Recombinant adeno-associated virus 2 ; Site-specific integration ; Transduction
• 刊名：Electronic Journal of Biotechnology
• 出版年：2016
• 出版时间：July 2016
• 年：2016
• 卷：22
• 期：Complete
• 页码：75-80
• 全文大小：753 K

文摘

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 × 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 72 h 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 180 d of cultivation.

Conclusions

A HEK293T cell line able to site-specifically integrate and stably express GDNF was established.