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Mol. Cells 2012; 33(5): 449-455

Published online April 17, 2012

https://doi.org/10.1007/s10059-012-2167-7

© The Korean Society for Molecular and Cellular Biology

Efficient Enhancement of Lentiviral Transduction Efficiency in Murine Spermatogonial Stem Cells

Bang-Jin Kim1, Ki-Jung Kim1, Yong-Hee Kim1, Yong-An Lee1, Byung-Gak Kim1,4, Chul Min Cho2,5,
Hye-Ryeon Kang1, Chul Geun Kim3, and Buom-Yong Ryu1,*

1Department of Animal Science and Technology, Chung-Ang University, Ansung 456-756, Korea, 2BET Research Institute, Chung-Ang University, Ansung 456-756, Korea, 3Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791, Korea, 4Present address: Department of Pharmacology, University of Pennsylvania Medical School, Philadelphia, USA, 5Present address: Department of Neurology and Neurosurgery, Lady Davis Institute, McGill University, Montreal, Canada

Correspondence to : *Correspondence: byryu@cau.ac.kr

Received: August 16, 2011; Revised: March 13, 2012; Accepted: March 14, 2012

Abstract

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout postnatal life in male and have the ability to transmit genetic information to the subsequent generation. In this study, we have optimized the transduction efficiency of SSCs using a lentiviral vector by considering different multiplicity of infection (MOI), dura-tion of infection, presence or absence of feeder layer and polycationic agents. We tested MOI of 5, 10 or 20 and infection duration of 6, 9 or 12 h respectively. After infec-tion, cells were cultured for 1 week and as a result, the number of transduced SSCs increased significantly for MOI of 5 and 10 with 6 h of infection. When the same con-dition (MOI of 5 with 6 hours) was applied in presence or absence of STO feeder layer and infected SSCs were cultured for 3 weeks on the STO feeder layer, a significant increase in the number of transduced cells was observed for without the feeder layer during infection. We subsequently studied the effects of polycationic agents, polybrene and dioctadecylamidoglycyl spermine (DOGS), on the transduction efficiency. Compared with the polybrene treatment, the recovery rate of the transduced SSCs was significantly higher for the DOGS treatment. Therefore, our optimization study could contribute to the enhancement of germ-line modification of SSCs using lentiviral vectors and in generation of transgenic animals.

Keywords germline modification, Lentivirus, polycationic agent, spermatogonial stem cell, transgenesis

Article

Research Article

Mol. Cells 2012; 33(5): 449-455

Published online May 31, 2012 https://doi.org/10.1007/s10059-012-2167-7

Copyright © The Korean Society for Molecular and Cellular Biology.

Efficient Enhancement of Lentiviral Transduction Efficiency in Murine Spermatogonial Stem Cells

Bang-Jin Kim1, Ki-Jung Kim1, Yong-Hee Kim1, Yong-An Lee1, Byung-Gak Kim1,4, Chul Min Cho2,5,
Hye-Ryeon Kang1, Chul Geun Kim3, and Buom-Yong Ryu1,*

1Department of Animal Science and Technology, Chung-Ang University, Ansung 456-756, Korea, 2BET Research Institute, Chung-Ang University, Ansung 456-756, Korea, 3Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791, Korea, 4Present address: Department of Pharmacology, University of Pennsylvania Medical School, Philadelphia, USA, 5Present address: Department of Neurology and Neurosurgery, Lady Davis Institute, McGill University, Montreal, Canada

Correspondence to:*Correspondence: byryu@cau.ac.kr

Received: August 16, 2011; Revised: March 13, 2012; Accepted: March 14, 2012

Abstract

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout postnatal life in male and have the ability to transmit genetic information to the subsequent generation. In this study, we have optimized the transduction efficiency of SSCs using a lentiviral vector by considering different multiplicity of infection (MOI), dura-tion of infection, presence or absence of feeder layer and polycationic agents. We tested MOI of 5, 10 or 20 and infection duration of 6, 9 or 12 h respectively. After infec-tion, cells were cultured for 1 week and as a result, the number of transduced SSCs increased significantly for MOI of 5 and 10 with 6 h of infection. When the same con-dition (MOI of 5 with 6 hours) was applied in presence or absence of STO feeder layer and infected SSCs were cultured for 3 weeks on the STO feeder layer, a significant increase in the number of transduced cells was observed for without the feeder layer during infection. We subsequently studied the effects of polycationic agents, polybrene and dioctadecylamidoglycyl spermine (DOGS), on the transduction efficiency. Compared with the polybrene treatment, the recovery rate of the transduced SSCs was significantly higher for the DOGS treatment. Therefore, our optimization study could contribute to the enhancement of germ-line modification of SSCs using lentiviral vectors and in generation of transgenic animals.

Keywords: germline modification, Lentivirus, polycationic agent, spermatogonial stem cell, transgenesis

Mol. Cells
Jan 31, 2023 Vol.46 No.1, pp. 1~67
COVER PICTURE
RNAs form diverse shapes and play multiple functions as central molecules of gene expression. In this special issue on RNA, seven minireviews illustrate how basic concepts and recent RNA biology findings are transformed into new and exciting RNA therapeutics.

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