160 Identification of bovine myostatin core promoter and its application for transgenesis in vitro

As a major regulator of myogenesis, myostatin expression has been of interest in agricultural and medical fields. The structure of myostatin promoter have been reviewed and interspecies conservation was highlighted. Therefore, it was postulated that there could be a core region of the myostatin (MSTN) promoter, and it could serve as a promoter for transgenesis. We cloned the whole (3 kbps) and fragmented bovine MSTN promoter, with green fluorescent protein (GFP) open reading frame (ORF) at 3′ of promoter, into plasmids with the piggyBac transposon system. Starting from ORF to 5′, sequences containing enhancer box (E box) 1 and 2 were grouped as Fragment 1 (243 bp); E box 3, 4, 5, 6 were Fragment 2; and E box 7, 8, 9, 10 (538 bp) were Fragment 3 (571 bp). These vectors with different promoter were delivered with transposase vector to ensure integration of cloned vectors into recipient genome. The concentration of piggyBac vector and transposase vector was 50 ng μL⁻¹ throughout all experiments. Given the biological significance, cloned vectors were transfected into the C2C12 myoblast cell line first. After transfection, expression levels of GFP were measured by quantitative real-time PCR. Compared with the whole promoter, GFP expression level was 6-fold higher with fragment #1, while expression levels were halved and barely seen by fluorescence microscopy with other fragments. When differentiated into myotubes, expression levels were increased 1.5-fold to 2-fold. Fragment #1 showed highest expression, but also showed the smallest change in expression during differentiation. In this step, we supposed fragment #1 is the core promoter of bovine MSTN promoter and modified it to make it shorter or stronger. The modifications included removing or duplicating 73 bp after the 1st TATA box and attaching enhancer sequence from CMV promoter to 5′ of fragment #1. Then the vectors were transfected into 10 different cell lines: C2C12, 3T3-L1, C127:LT, MCF-7, Hela, A549, PC-3, Cal-62, MDBK, and immortalized bovine fibroblast. After transfection, mean fluorescence intensity (MFI) was measured for each group by flow cytometry. For fragment #1, MFI ranged 4388 to 44 348, according to cell types. Modifications with 73 bp after TATA box did not show improvements; MFI ranged from 635 to 5274 when 73 bp was removed, and 4867 to 36 031 when duplicated. However, when CMV enhancer were attached, fluorescence was increased with MFI from 6611 to 72 816. Based on cell-based results, fragment #1 vector was directly microinjected into the presumptive bovine zygote 16 h post-fertilization. During 4 times of microinjection, a total of 272 zygotes were included, 35.0 ± 2.7% of embryos cleaved into 8-cell, and 7.8 ± 5.2% reached blastocyst at day 8 post-fertilization. Among embryos cleaved, 31.9 ± 13.9% showed GFP and 59.6 ± 25.9% of blastocysts showed GFP. In conclusion, we suggest proximal 243 bp of MSTN promoter as core region and its potential application to induce transgene into cell and embryo.

This work was supported by the Technology Innovation Program (20023353) funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea).