Increase in the titer of lentiviral vectors expressing potassium channels by current blockade during viral vector production.
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 Ba(2+) 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 Ba(2+) 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.


CONCLUSIONS
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.
Department of Physiology, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka, 573-1010, Japan. mokada-tky@umin.ac.jp.
Masayoshi
M
Okada
Department of Physiology, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka, 573-1010, Japan. naaz.andharia@yahoo.com.
Naaz
N
Andharia
Department of Physiology, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka, 573-1010, Japan. matsudah@hirakata.kmu.ac.jp.
Hiroko
H
Matsuda
D000818:Animals; D001464:Barium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism; D057809:HEK293 Cells; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D051665:Kv1.4 Potassium Channel / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000494:pharmacology; D024661:Potassium Channels, Inwardly Rectifying / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D024683:Potassium Channels, Tandem Pore Domain / Q000235:genetics / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D016704:Synapsins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D019562:Viral Load
D002413:Cations, Divalent; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; C489461:Kir2.1 channel; D051665:Kv1.4 Potassium Channel; D026902:Potassium Channel Blockers; D024661:Potassium Channels, Inwardly Rectifying; D024683:Potassium Channels, Tandem Pore Domain; D016704:Synapsins; C103742:potassium channel protein TREK-1; D001464:Barium; D011188:Potassium