[Regulation of AAV-mediated glial cell-line derived neurotrophic factor expression by using improved Tet-on
Teng Chen1, Xin-Gang Li, Cheng-Yuan Wu
1Department of Neurosurgery, Qilu Hospital, Shandong University, Jinan 250012, China.
Objective:
To control the expression of AAV-mediated glial cell-line derived neurotrophic factor (GDNF) gene purposely by incorporating novel Tet-On trans-activator rtTA2s-S2, which prevents potential harms caused by over-expression of recombinant target genes.
Methods:
Oligonucleotide with specific monocloning sites was inserted into the hHG part of pAAV-GDNFflag. Trans-activator from pUHrT61-rtTA2s-S2 and TRE from pTRE-d2EGFP were amplified by PCR and inserted into pCRII-TOPO respectively. Possible mutation was eliminated by sequencing. TRE and rtTA2s-S2 were then inserted into the oligonucleotide of pAAV-GDNFflag to form pAAV-rtTA2s-S2-TRE-GDNFflag. pAAV-rtTA2s-S2-TRE-d2EGFP was constructed by replacing the GDNFflag part with d2EGFP. The plasmids were digested by Xba I and compared with theoretic values. HEK 293 cells were cultured and co-transfected with pAAV-GDNFflag and helper plasmids pHLP19 and pAdeno5 so as to complete the package of recombinant adeno-associated virus (rAAV) Crude virus lysate was purified by two-sequential continuous CsCl gradient ultracentrifugation, dialyzed and condensed by millipore filter. The titer of rAAV was determined by real-time quantitative PCR. Another HEK293 cells were cultured, transfected with rAVV, and then cultured in 2 kinds of culture fluid: with or without doxycyclin (Dox). Fluorescence microscopy was used to calculate the percentage of fluorescent cells so as to detect AAV-rtTA2s-S2-TRE-d2EGFP, and Western blotting was used to detect the GDNF protein in the lysate of the HEK293 cells, thus testifying the regulatory function in vitro of rtTA2s-S2. Twenty male Wistar mice were randomly divided into 2 groups: experiment group, fed with Dox and sucrose (Dox-positive group), and control group, fed with only sucrose (Dox-negative group). Two days after AAV-rtTA2s-S2-TRE-GDNF was injected into the gastrocnemius muscles of the mice. Two weeks the mice were killed and their gastrocnemius muscles were taken out. ELISA was used to examine the content of GDNF in the homogenate of gastrocnemius muscle so as to examine the regulatory effect of rtTA2s-S2 in vivo.
Results:
Tests showed that the recombinant plasmids were constructed correctly. The fluorescent cell positive rate in the Dox-positive culture fluid of HEK293 cells was 52.4%, significantly higher than that in the Dox-negate culture fluid (7.2%, P < 0.01). Western blotting of the HEK 293 cell lysate showed clear band of GDNF in the Dox-positive group and failed to show visible band in the Dox-negative group. The content of GDNF in the homogenate of gastrocnemius muscles of the Dox-positive group was 32.6pg/ml +/- 2.6 pg/ml, significantly higher than that of the Dox-negative group (10.1 pg/ml +/- 2.4 pg/ml, P < 0.01).
Conclusion:
Novel Tet-on trans-activator rtTA2s-S2 regulates downstream AAV-mediated GDNF expression in a stringent manner and does not impair AAV infecting efficiency when constructed together with TRE and GDNF within one AAV vector.
