Increase in the titer of lentiviral vectors expressing potassium channels by current blockade during viral vector production

BMC Neuroscience

Hiroko Matsuda¹  Naaz Andharia¹  Masayoshi Okada¹ 

[1] Department of Physiology, Kansai Medical University, 2-5-1 Shin-machi, Hirakata 573-1010, Osaka, Japan

关键词: HERG;    Kv1. 4;    TREK-1;    Kir2. 1;    Method improvement;    K+ channel;    Titer;    Lentiviral vector;   

Others  :  1211791
DOI  :  10.1186/s12868-015-0159-1

 received in 2015-02-18, accepted in 2015-04-01,  发布年份 2015

【 摘 要 】

Background

High titers of lentiviral vectors are required for the efficient transduction of a gene of interest. During preparation of lentiviral the vectors, the protein of interest is inevitably expressed in the viral vector-producing cells. This expression may affect the production of the lentiviral vector.

Methods

We prepared lentiviral vectors expressing inwardly rectifying potassium channel (Lv-Kir2.1), its dominant-negative form (Lv-Kir-DN), and other K⁺ channels, using the ubiquitously active β-actin and neuron-specific synapsin I promoters.

Results

The titer of Lv-Kir-DN was higher than that of Lv-Kir2.1, suggesting a negative effect of induced K⁺ currents on viral titer. We then blocked Kir2.1 currents with the selective blocker Ba²⁺ during Lv-Kir2.1 production, and obtained about a 5-fold increase in the titer. Higher extracellular K⁺ concentrations increased the titer of Lv-Kir2.1 about 9-fold. With a synapsin I promoter Ba²⁺ increased the titer because of the moderate expression of Kir2.1 channel. Channel blockade also increased the titers of the lentivirus expressing Kv1.4 and TREK channels, but not HERG. The increase in titer correlated with the K⁺ currents generated by the channels expressed.

Conclusion

In the production of lentivirus expressing K⁺ channels, titers are increased by blocking K⁺ currents in the virus-producing cells. This identifies a crucial issue in the production of viruses expressing membrane channels, and should facilitate basic and gene therapeutic research on channelopathies.

【 授权许可】

   
2015 Okada et al.; licensee BioMed Central.

【 图 表 】

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