Transfiction of Holstein Bull Spermatozoa by Lipofectamine

Introduction A simple and efficient method for producing multi-transgenic animals is required for medical and veterinary applications. The principal technique for the production of transgenic animals is pronuclear microinjection, which has a low efficiency for the generation of transgenic farm animals expressing a single transgene. Recently, nuclear transfer has been used to clone large animals, and could allow multiple genetic manipulations to be undertaken in vitro, prior to a single nuclear transfer, rather than complex and time consuming breeding programs. However, at present the frequency of success in cloning large animals is very low and is very expensive. The production of transgenic livestock by sperm mediated gene transfer (SMGT) has a number of advantages compared to other transgenic techniques such as nuclear transfer and microinjection. Both nuclear transfer and microinjection techniques are technically demanding and labor intensive. In contrast, SMGT requires no sophisticated equipment or technical expertise. Furthermore, bovine genetics are distributed through sperm in the dairy industry consequently making it easy to distribute genetically modified sperm. SMGT is based on the ability of sperm to bind to exogenous DNA molecules and transfer it to the oocyte during fertilization. The major benefits of the SMGT technique were found to be its high efficiency, low cost and ease of use compared to other methods. SMGT was first described in a small animal model, with high efficiency reported in the mouse. Recently the technique has been successfully adapted and optimized for use in large animals. Studies have shown that spermatozoa from numerous species, including bovine, can bind and take up foreign DNA and transfer it to the embryo. In bovine studies, the efficiency of SMGT can vary widely depending on both the transgene and the gene transfer method. Liposomes have been shown to be particularly effective in transferring DNA into bovine sperm. However, not all embryos derived from transfected sperm contain the transgene, suggesting that mechanisms exist, which impede SMGT. The aim of this study was to investigate the possibility of gene transfer to bull spermatozoa by using lipofection.
Materials and Methods Ram testes collected from Meysam abattoir slaughterhouse immediately after slaughter and were brought to the laboratory in an ice chest. In the laboratory, the testes were rinsed twice with normal saline and were then trimmed to remove the extra testicular tissue and washed properly with saline containing 0.1% streptomycin sulphate. Connective tissue covering the cauda epididymis was removed by careful dissection, with care to avoid rupturing blood vessels or the epididymal duct. For detection of transfected spermatozoa, they stained with Rodamine. In order to transfection of sperm, 2 μg of Rodamine labeld DNA and 0.5 μl of TurboFect were diluted in 25 μl of transfection medium separately, and incubated for 5 min at room temperature. Then, the diluted DNA was added to diluted TurboFect (total volume=50 μl) and incubated for 20 min at room temperature. 1×106 sperm were added to 50 μl of DNA- TurboFect complexes and mixed gently by rocking the plate back and forth. To evaluation of transfected spermatozoa motility, acridine orange staining was used. Each experiment was replicated at least three times, and for each replicate, at least 50 ES cell colonies were used. Data were analysed with a statistical software program (SPSS 16). Comparisons between two treatments and multiple numeric datasets were performed using t-test and one-way ANOVA followed by Duncan multiple-range test, respectively. Results are expressed as mean±SEM and statistical significance was accepted at P0.05). The comparison of transfected and normal spermatozoa reveal that, motility of transfected spermatozoa at 60 minutes after transfection was significantly lower than normal ones (p