Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector|
Zhang, Jinju; Zhang, Yun; Liu, Xiaomei; Xiang, Jingjing & Zhang, Chun
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 × 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.
Glial cell line-derived neurotrophic factor; Multiplicity of infection; Recombinant adeno-associated virus 2; Site-specific integration; Transduction