20 GENERATION AND CHARACTERIZATION OF TRANSGENIC-CLONED PIGS EXPRESSING THE FAR-RED FLUORESCENT PROTEIN MONOMERIC PLUM

Transgenic (Tg) pigs expressing a fluorescent protein are extremely useful for research into transplantation and regenerative medicine. This study aimed to create Tg pigs expressing monomeric Plum (mPlum), a far-red fluorescent protein with a longer wavelength than enhanced green fluorescent protein (EGFP) and humanized Kusabira Orange (huKO), the two fluorescent proteins that have been used previously for Tg pig production. A linearized CAG-mPlum transgene construct was transferred into porcine fetal fibroblasts (PFF) by electroporation. mPlum fluorescence-positive cells were collected using a cell sorter and used as nuclear donors (mPlum-PFF) for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus–oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. Then, SCNT was performed as reported previously (Matsunari et al., 2008). The reconstructed embryos were cultured for 7 days in porcine zygote medium-5 (PZM-5). mPlum fluorescence expression was screened during the early development of the embryos. After 5 or 6 days of culture, the SCNT embryos were surgically transferred to the uterus of a recipient gilt. We first obtained fetuses on Day 36 or 37 of gestation by Caesarean section and the PFF were retrieved from their skin. Fluorescence expression was analysed using fluorescence microscope, and the number of transgene copies in each fetus was determined by Southern blot analysis. We also analysed whether unique spectral properties of mPlum are suitable for multicolor imaging using confocal microscope and flow cytometer. The identification of mPlum-expressing PFF under the mixed culture of PFF expressing EGFP and huKO was examined. The 2 cell lines of PFF expressing EGFP and huKO were previously generated in our laboratory. Rates of normal cleavage and blastocyst formation occurred in the SCNT embryos generated with mPlum-PFF (mPlum embryos) were equivalent to those of SCNT embryos derived from nontransgenic PFF (34/42, 81.0%; 33/42, 78.6% v. 37/40, 92.5%; 30/40, 75.0%). Total cell numbers in mPlum and control blastocysts did not differ significantly (88.3 ± 6.0 v. 99.9 ± 8.8). Fluorescence expression in the mPlum embryos began at the 8-cell stage and became brighter from the morula stage. The gilt into which 103 mPlum embryos were transferred produced 3 fetuses. These fetuses expressed mPlum fluorescence systemically and had 1 to 5 copies of the transgene. Multicolor fluorescence imaging and flow cytometric analyses of a mixed culture of mPlum PFF and PFF expressing EGFP and huKO showed that clear identification and isolation of cells displaying each of the 3 fluorescence signals was possible. These observations demonstrate that the transfer of CAG-mPlum did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of Tg cloned pigs that systemically expressed mPlum. This work was supported by JSPS KAKENHI Grant Number 25293279.