Abstract
The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes.
Highlights
Methods that allow for the coordinated expression of multiple genes in eukaryotic cells have the potential to significantly enhance our ability to genetically engineer the porcine genome
Simultaneous introduction of multigene constructs using this method is problematic, because it is difficult to place multiple expression units into a single construct and, even if successful, the expression of one or more expression units is usually suppressed, most likely owing to gene silencing [2]
The aim of this study was to demonstrate the ability of this PB-based gene delivery system to allow simultaneous introduction of multigene constructs into the genome of porcine cells through a single transfection event
Summary
Methods that allow for the coordinated expression of multiple genes in eukaryotic cells have the potential to significantly enhance our ability to genetically engineer the porcine genome. Lavitrano and colleagues recently succeeded in introducing multiple transgenes into the pig genome using sperm-mediated gene transfer technology [3] They observed transgene expression in the resulting piglets. This technology is not widely used, probably because of the technical difficulties associated with the technique In this context of genetically engineered animals, it is preferable to employ the somatic cell nuclear transfer (SCNT) method, despite its very low efficiency [4]. The application of other pre-existing selectable markers would allow us to realize such a concept In this case, it is desirable to obtain transfectants carrying multigene constructs through a single transfection event, since multiple transfections cause deleterious effects on the normal function of a cell, which in turn causes frequent failures in the SCNT-mediated production of cloned piglets
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