Efficient Enhancement of Lentiviral Transduction Efficiency in Murine Spermatogonial Stem Cells

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), duration 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 infection, 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 condition (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.

Direct modification of spermatogonial stem cells using lentivirus vectors in vivo leads to efficient generation of transgenic rats

“…The polycationic agents polybrene and DOGS are often used to increase the transduction efficiency in SSCs in vitro 17. However, they are cytotoxic following virus infection.…”

“…To assess the toxicity of polycationic agents on the testis, 4 μg ml −1 of polybrene (Sigma-Aldrich, St. Louis, MO, USA) and 2 μg ml −1 of dioctadecylamidoglycyl spermine (DOGS; Promega, Madison, WI, USA) were dissolved in Dulbecco's phosphate-buffered saline (PBS) and approximately 20 μl was injected into the seminiferous tubule of the testes of 3-week-old rats. The concentrations of polybrene and DOGS were chosen based on our previous study 17…”

“…Several groups have reported the development of transgenic rats using the conventional lentivirus-mediated SSC transduction and transplantation technique 910. To refine transduction conditions, we previously evaluated the transduction efficiency of mouse SSCs using various durations of lentiviral treatment, various multiplicities of infection and addition of different concentrations and types of polycationic agents 17. As a model for human disease, rats have many advantages over mice and other animals.…”

A standard procedure for lentiviral‐mediated labeling of murine mesenchymal stromal cells in vitro

“…Manipulation of gene expression holds great promise in the biomedical research field. Among the large collection of devices developed for gene transferring to various cells , the lentiviral vectors appeared as one of the most powerful tools , due to their high transduction efficiency, stable transgene integration and expression , and low immunogenicity . They can be obtained on either transient or continuous production platforms.…”

Polybrene: Observations on cochlear hair cell necrosis and minimal lentiviral transduction of cochlear hair cells

“…Polybrene (hexadimethrine bromide), a cationic polymer, is widely used to enhance retroviral and lentiviral transduction of various cell types, including mouse mesenchymal stem cell lines, cutaneous cells, human keratinocyte stem cells, and murine spermatogonial stem cells [7,10,14,20]. Polybrene bridges the virus to the cell membrane, eliminating negative charges, thus facilitating viral absorption to the cell surface [8].…”