Application of piggyBac transposon system in transgenic pig cloning

Abstract

PiggyBac transposon has been widely employed to generation transgenic animals using a "cut and paste" mechanism that is occurred by piggyBac transposase recognizing transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of targeted gene. The targeted gene is cut, and then transferred in to TTAA chromosomal sites. In this study, to investigate whether this system can be applied to transgenic cell line establishment, generation of cloned embryos by somatic cell nuclear transfer (SCNT) and also trial of re-differentiation of cell that was generated by transcription factor for donor in producing transgenic pig SCNT cloned embryos. Firstly, to verify the application of piggyBac transposon to establish transgenic cell line and generate its cloned embryos by SCNT, a report gene, RFP (DsRed2), inducible expression vector was constructed using piggyBac transposon system, it was transferred to porcine fetal fibroblast, after enrichment, the transgenic cell line was established. Homogenously RFP expression was well controlled according the fact of existence or nonexistence of doxycycline in established cell line. Also cloned embryos were shown homogenous expression pattern of RFP using the cell line for SCNT donor. Furthermore the cloned embryos were shown normal development without damage by piggyBac transposon system. Octamer-binding transcription factor 4 (Oct4) is a critical molecule for the self-renewal and pluripotency in undifferentiated cell lines like as embryonic stem cell. For investigate the effect of Oct4-overexpression on cell proliferation and development of cloned embryos, Oct4 was firstly transferred to porcine fibroblasts before the introduction of 4 transcription factors in further. Oct4 expression was validated by the immunostaining of Oct4. Cell morphology was changed to sharp, and both proliferation and migration abilities were enhanced in Oct4-overexpressed cells and p16, Bcl2 and Myc were also upregulated. SCNT was performed using Oct4-overexpressed cells, and the development of Oct4 embryos was compared to that of wild-type cloned embryos. The cleavage and blastocyst formation rates were significantly improved in the Oct4 embryos. Interestingly, blastocyst formation of the Oct4 embryos was observed as early as Day 5 in culture, while blastocysts were observed from Day 6 in wild-type cloned embryos. For validating the availability of piggyBac transposon application to generate porcine induced pluripotent cells, coding domain region of porcine Oct4, Sox2, c-Myc and Klf4 from pig ovarian cDNA by PCR was cloned, constructed to piggyBac expression vector, which is inducible expressed by doxycycline and transduced to porcine fetal fibroblast. After 24h transfection, 2 µg/ml doxycycline was added in DMEM/F12 culture media contained 15% FBS, 10ng/ml bFGF and transfected cells were cultured for 2 weeks more. From about 10 days after, outgrowing colonies were formed, picked, digested to single cell and cultured on CF1 feeder cells treated with mitomycin C. They are routinely single cell passaged 1:4~6 at every 2-3 day by enzyme. We verified the colony formation from single cell culture and maintained the colonies up to passage (> 37th). To compare the developmental efficiency of differentiated cells and established cell lines. SCNT was carried out using two types of cells as followed to our previous established protocol. In the results, the cleavage rate and blastocyst formation rate were not shown any significant differences. But total cell number of blastocyst that one of parameter evaluating of quality of embryos was shown significantly increased in established cell lines group. In conclusion, piggyBac transposon system could be applied to establish the transgenic cell lines and cloned embryos for producing of transgenic pig, also it could be used to generated illimitable self-renewal cell line and produce the its cloned embryos. As this gene delivery system will be valuably used in gene modification availability and stability or safety of generating pig models for human biomedical research.