Abstract
Sperm-mediated gene transfer can be a very efficient method to produce transgenic pigs, however, the results from different laboratories had not been widely repeated. Genomic integration of transgene by injection of pseudotyped lentivirus to the perivitelline space has been proved to be a reliable route to generate transgenic animals. To test whether transgene in the lentivirus can be delivered by sperm, we studied incubation of pseudotyped lentiviruses and sperm before insemination. After incubation with pig spermatozoa, 62±3 lentiviral particles were detected per 100 sperm cells using quantitative real-time RT-PCR. The association of lentivirus with sperm was further confirmed by electron microscopy. The sperm incubated with lentiviral particles were artificially inseminated into pigs. Of the 59 piglets born from inseminated 5 sows, 6 piglets (10.17%) carried the transgene based on the PCR identification. Foreign gene and EGFP was successfully detected in ear tissue biopsies from two PCR-positive pigs, revealed via in situ hybridization and immunohistochemistry. Offspring of one PCR-positive boar with normal sows showed PCR-positive. Two PCR-positive founders and offsprings of PCR-positive boar were further identified by Southern-blot analysis, out of which the two founders and two offsprings were positive in Southern blotting, strongly indicating integration of foreign gene into genome. The results indicate that incubation of sperm with pseudotyped lentiviruses can incorporated with sperm-mediated gene transfer to produce transgenic pigs with improved efficiency.
Highlights
In 1989, two independent reports claimed that sperm cells could associate with exogenous DNA molecules and transfer of these molecules during fertilization, resulting in genetically modified offspring [1,2]
By RT-PCR amplification of the RNA specific sequence of the lentivirus absorbed by sperm, the results showed that the lentiviral mRNA was present in the incubated sperm after several times of washing (Figure 1)
Ct value was plotted against known lentiviral number to draw the standard curve (y = 20.207*Log (x)+27.04) (Figure S1), Lentiviral particles was calculated with Ct value
Summary
In 1989, two independent reports claimed that sperm cells could associate with exogenous DNA molecules and transfer of these molecules during fertilization, resulting in genetically modified offspring [1,2] This process, termed sperm-mediated gene transfer (SMGT), provides a simple and straightforward method to produce transgenic animals. The foreign DNA would be transmitted to the generation by being maintained as an extrachromosomal structure (episome) in the positive transgenic animals [14,15]. This may account for non-stable inheritance in SMGT with plasmid DNA [8,16,17]. SMGT has not been widely adopted for making transgenic animals
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