Studies On Expression And Construction Of Mammary Gland Expression Vector For Human Lactoferrin Gene Mediated By The Sleeping Beauty Transposon System

The Sleeping Beauty (SB) transposon system, a member of the Tel/mariner super family of "cut-and-paste" transposable elements, is reconstructed from the salmon fish genome by restoring its activity through the correction of accumulated mutations and is developed as a non-autonomous transposon system. It consists of two parts:The SB transposon which is excised from the genome and reinserted into other locations of the genome, and the enzymatic factor that catalyzes the transposition reaction, namely the transposase. The Sleeping Beauty (SB) transposon system is found to show the most efficient transposition in vertebrate cells and is utilized as a tool for germ line mutagenesis and gene delivery.Human lactoferrin (hLF), an iron-binding glycoprotein of about80kDa of the transferrin family, exists in mucosa and most body fluids, has a broad spectrum of antimicrobial properties. However, production of human lactoferrin purified by purification from human milk safety potential safety, and supply limited. And supply limitations. Therefore, several transgenic approaches have been adopted to produce human lactoferrin.In this study, a mammary gland expression vector with β-lactoglobulin (BLG) as regulatory region and SB transposon system as gene delivery system was constructed; the double marker genes of neo and EGFP were sub-cloned into the vector, and were transfected into the caprine ear fibroblast cells that were cultivated in the conditional medium created. This shortened the time for selecting by G418and facilitated the selection of modified cells by EGFP gene. We attempted to establish an effective and reliable procedure to prepare transgenic cells which could be used for the production of transgenic mammary gland bioreactors by SCNT. The concrete contents of our experiment are as follows:(1) Cloning and identification of the β-lactoglobulin gene Designed and synthesized the primers of5’BGL.3’BLG and EGFP,4.2kb5’BGL and1.2kb3’BLG were amplified by PCR from goat genomic DNA, and0.8kb EGFP was amplified by PCR from pEGFP-C1. pcDNA3.1(-)-BLG-EGFP vector was constructed by cloning three fragments (5’BLG,3’BLG and EGFP) into the same restriction sites of pcDNA3.1(-)with T4DNA ligase. The purified vector was transfected into caprine mammary epithelial cells (CMECs), which were cultured in the medium with hormones. The expression of EGFP reporter gene in transgenic CMECs revealed that the5’BLG and3’BLG could promote the expression of reporter gene.(2) Construction and identification of hLF mammary gland expression vector mediated by the SB transposon system2.1kb hLF was amplified by PCR from cDNA of hLF, the EGFP was replaced with hLF, and it was cloned into the same restriction sites of pT2, then the mammary gland expression vector pT2-BLG-hLF was constructed. To ensure the correctness of the construction, the vector was identified by PCR amplification and restriction enzymatic digestion. Therefore, the caprine mammary epithelial cells (CMECs) were induced by stimulation with hormone signals (ITS, EGF, hydrocortisone and prolactin) and employed to examine the availability of hLF transgenic expression in vitro. Then, the purified plamid and the SB transposase were transfected into the cells. The recombinant hLF characteristic expression was detected by Western-Blotting from the supernatant of transfected CMECs, and expression of mRNA was determined by RT-PCR. The result confirmed that the pT2-BLG-hLF possessed the desirable bioactivity to efficiently express hLF in CMECs. Paramount to everything, this study laid a firm foundation for preparing the hLF gene transgenic goat fetal-derived fibroblast cells.(3) Generation of caprine ear fibroblast cell line stablely integrated with hLFIn this study, a mammary gland expression vector pT2-BLG-hLF-EGFP-Neo, with BLG as regulatory region、hLF as target gene、SB transposon system as gene delivery was constructed, the double marker genes of neo and EGFP were sub-cloned into the vector. The purified plamid and SB transposase were transfected into the caprine ear fibroblast cells. After being screened by700μg/mL of G418for10-14d, the colonies were formed. A single clone was detached by rings for picking clones and reseeded in the24-well plates under G418selection. Genomic DNA of G418-resistant cells was extractedand analyzed by PCR amplification. This research may provide an effective upstream system to prepare SCNT donor cells for the production of human recombinant pharmaceuticals from the milk of transgenic animals.