Testis-mediated gene transfer in mice: comparison of transfection reagents regarding transgene transmission and testicular damage

Abstract

Testis-mediated gene transfer (TMGT) has been used as in vivo gene transfer technology to introduce foreign DNA directly into testes, allowing mass gene transfer to offspring via mating. In this study, we used plasmid DNA (pEGFP-N1) mixed with dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMA) or liposome (Lipofectin) in an attempt to improve TMGT. Males receiving consecutive DNA complex injections were mated to normal females to obtain F0 progeny. In vivo evaluation of EGFP expression, RT-PCR and PCR were used to detect the expression and the presence of exogenous DNA in the progeny. We also evaluated possible testicular damage by histological procedures. PCR and RT-PCR analyses revealed that liposome and DMSO increased the rate of TMGT. Histological analyses demonstrated that repeated (4 times) injections of DNA complexes can affect spermatogenesis. DMSO was the most deleterious among the reagents tested. In this study, we detected the presence of transgene in the progeny, and its expression in blood cells. Consecutive injections of DNA complexes were associated with impaired spermatogenesis, suggesting requirement of optimal conditions for DNA delivery through TMGT.

Bacterial magnetic particles improve testes-mediated transgene efficiency in mice

“…However, Sato and Nakamura indicated that repeated injections (3 and 6 repeated injections, 3 days apart) were not critical for introducing high copy numbers of DNA into offspring (Sato & Nakamura, 2004). Furthermore, although DNA-DMSO complex could produce as many as 61%, 55%, and 80% of transgenic founders in mouse and/or rabbit through testes injection, DMSO-containing solution induces testicular degeneration and reduces vascularization around seminiferous tubules (Shen et al, 2006;Amaral et al, 2011). Notably, the efficiency of producing transgenic founder mice through one injection procedure performed in this study by applying BMPs was 88%, which was higher than existed reports.…”

“…However, the most popular methods in producing such animals remains low efficiency, which either requires high technologies, expensive instruments, has safety risks or brings adverse effects upon cells and tissues (Smith, 2004;Parrington et al, 2011), and as a result, are not easily handled by individual laboratories. Previously developed spermatozoa-based gene transfer methods, including testis-and sperm-mediated gene transfer (TMGT and SMGT) can be performed by general researchers to achieve personalized gene modifications in animals (Coward et al, 2007;Collares et al, 2010;Amaral et al, 2011;Campos et al, 2011a,b). Referring to TMGT, foreign DNA vehicles need to be excellent in penetrating thick tissues to achieve better output.…”

Down Regulation of Catsper1 Expression by Calmodulin Inhibitor (Calmidazolium): Possible Implications for Fertility

“…It has been reported in the literature that DMSO acts as a free-radical scavenger against the effects of radiation and helps preserve some male fertility [ 24 ]. Possible causes of impaired male fertility could be the number of injections in testicles and cell damage, since DMSO with DNA used as a transfection agent causes marked histological changes in seminiferous tubules [ 18 ].…”

“…Both transcription factors have a synergistic effect, revealing a central role for Sox in the Catsper1 gene transcription [ 16 ]. The Catsper1 promoter has not been evaluated in the testicular environment [ 17 , 18 , 19 , 20 , 21 , 22 ], where Sox factors could affect its activity as they do in vitro.…”

Possible Production of Genome-Edited Animals Using Gene-Engineered Sperm

“…On the contrary, He et al [53] demonstrated that the transgene transmission rate from F0 to F1 generation was 37%, suggesting that the transgene transmission rate was similar to the Mendelian law of inheritance. In 2007 and onward, attempts to improve IIGT systems were made by several laboratories to enhance the gene delivery efficiency [54][55][56][57][58][59][60][61][62][63][64][65][66][67]. In the following sections, we will describe several examples [(v) to (x)] about the improvement of IIGT, in vitro assessment for gene expression after IIGT or possible mechanism underlying IIGT.…”