The Application Of Cell-penetrating Peptides Technology In The Pig Genome Modification And The Produce Of The Marker Gene Self-excision Pigs

Cell-penetrating peptides (CPPs) are a kind of peptides with a maximum of 30 amino acids.They can deliver a large variety of cargoes into the cell membrane, including plasmid DNA, oligonucleotides, small interfering RNAs (siRNAs), peptides and protein. Cell-penetrating peptides (CPPs) are efficient as a transport tool, and they are non-toxic and easy to operate. So far, it had been successfully used both in vitro and in vivo. However, there is no such report in pig primary cells. The basic research and biomedical application of transgenic pigs will increasingly depend on the availability of technologies for efficient genetic modification of pig primary cells. Now the pig primary cell genome editing technology has many defects, such as, low efficiency, high cell toxicity, and complicate operation and so on. The marker gene which the transgenic pigs carried can misregulate both the adjacent genes and the gene of interest, it may also overstate the public about biological safety. Meanwhile, both the homozygous knockout and conditional transgenic technology in transgenic pig are lagging. These technical defects greatly limited the applications of transgenic pigs. Therefore, the development of new transgene technology for solving these problems is very important.In order to investigate whether the cell-penetrating peptides can uptake "cargo" into the pig primary cells and subsequently perform function, we firstly chose the three efficient cell-penetrating peptides (CPPs) including TAT, R9 and CPP5 which are widely investigated. We constructed three prokaryotic expression vectors pTAT-Cre, pR9-Cre and pCPP5-Cre for protein purification. In order to identify the activity of the purified protein which can be used in subsequent studies, in vitro recombination assay and double-fluorescence reporter cells assay were performed. These three cell-penetrating peptides can efficiently uptake the Cre recombinase into pig primary cells with a concentration-dependent manner in the Alexa 488 fluorescence labeling assay. The Maximum efficiency was 95%.In order to know whether the Cre recombinase transported to pig primary cells, which mediated by TAT, R9 and CPP5, can perform recombination function or not, and in order to test the recombination efficiency. The standard curve analysis method was used. We found that CPP5-Cre had 95% recombination efficiency at a high concentration, but recombination efficiency of the TAT-Cre and R9-Cre were lower than that of CPP5-Cre. In order to improve the recombination efficiency, we investigated the mechanism of internalization of the three Cre recombinant proteins in pig fetal fibroblasts. We found that proteins transduction was temperature dependent and end endosomal escape dependent in pig fetal fibroblasts by the Alexa 488 fluorescence labeling assay and endosome colocalized assay. Based on the research of this mechanism, HA2 (The N-terminal 20 amino acids of the influenza virus hemagglutinin protein) and chloroquine were used to improve the TAT-Cre recombination efficiency. And the results showed HA2 can improve the recombination efficiency of TAT-Cre to 90%.Considering future applications of these fusion recombinant proteins and small molecules in pig primary cells, we studied their cytotoxicity. Both three proteins and HA2 did not show significant cytotoxic effects even at 10 uM. However, the concentration of chloroquine (≥100 μM) was associated with high cytotoxicity in pig fetal fibroblasts. Subsequently, we successfully used TAT-Cre to produce the antibiotic selectable marker free knockout pigs.I also created a versatile self-excision system to improve transgene technology of large animals. This system based on conditional transgenesis can produce marker free and homozygous knockout pigs simply, fast and efficiently. Oct-4 is a gatekeeper in the beginning of mammalian development which is very important to maintain the mES cells and the induced pluripotent stem cells (iPSCs) pluripotency. It specially expressed in mouse preimplantation embryo. This system included an important element named OCN which takes advantage of embryo-specific promoter to drive expression of the Cre recombinase. The marker gene, linked to Cre, survived selection in pig primary cultured cells. But the marker gene removed along with Cre as both on the preimplantation embryo period. We constructed a MSTN gene targeting vector including the OCN element to target the pig primary cells. Oct4 cannot drive the Cre recombinase expression in cell culture, so the marker gene can use as a positive selection. Gene targeting cells were generated and used as donor cells for SCNT. Oct4 can drive the Cre recombinase expression to remove the marker gene in embryos. We produced the Fo marker free knockout pig successfully and efficiently. Meanwhile, marker free and homozygous knockout pigs were also produced efficiently based on this method.In summary, we, for the first time, expanded the PTD technology research in pigs and obtained marker free pigs using the PTD technology. Meanwhile, we established a mOct4 promoter drive Cre recombinase expression-based self-excision system. Both of the marker free knock out pigs and homozygous knockout pigs were generated simply, fast and efficiently. This study solved the biosafety problem and other defects of the large animal transgenic technology, it will promote the application of transgenic pigs in basic research and human disease models.