303 ESTABLISHMENT OF PREGNANCIES WITH HANDMADE CLONING PORCINE EMBRYOS RECONSTRUCTED WITH FIBROBLASTS CONTAINING AN ALZHEIMER'S DISEASE GENE

Abstract

Somatic cell nuclear transfer (SCNT) offers the possibility of pig transgenesis. Importantly, specific genetic manipulations can be performed in donor cells before SCNT to derive pig models for specific human genetic diseases, including the neurodegenerative disorder Alzheimer's disease (AD). In the present study, we established pregnancies after transfer of SCNT blastocysts produced by the handmade cloning (HMC) technique. The blastocysts were transgenic for a human gene, amyloid precursor protein gene with the 'Swedish mutation' (APPsw), causing AD. For transgenesis, minipig fibroblasts were transfected by lipofection with a vector containing the APPsw gene under control of the platelet-derived growth factor β promoter (PDGF-APPsw) and a neomycin-resistance selection gene. Neomycin-resistant colonies were isolated, expanded, analyzed, and used for HMC. Cumulus–oocyte complexes were aspirated from ovaries of slaughtered sows and matured for 41 h. Subsequently, the cumulus cells were removed in hyaluronidase, and zonae pellucidae were partially digested by incubation in pronase. Oocytes with a visible polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm adjacent to the PB was removed with a microblade. The cytoplasts were used as recipients for embryo reconstruction. Reconstructed embryos were produced by a 2-step fusion procedure. At the first step, 1 cytoplast was fused with 1 fibroblast in the absence of Ca²⁺. After 1 h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously in the presence of Ca²⁺, incubated in cytochalasin B and cycloheximide for 4 h, and then cultured in PZM-3 medium. The development of reconstructed embryos to the blastocysts stage was determined after 5, 6, or 7 days of in vitro culture. To investigate the in vivo developmental capacity, blastocysts were transferred surgically to synchronized recipients. When using PDGF-APPsw-transgenic minipig fibroblasts, the rate of blastocyst formation (mean ± SEM) was 39 ± 3% (164/424). In comparison, non-transgenic fibroblasts resulted in a blastocyst development of 36 ± 7% (36/102). In 4 recipients that received an average of 54 Day 5, 6, and 7 PDGF-APPsw-transgenic blastocysts, 2 ongoing pregnancies were confirmed by ultrasonography, 1 pregnancy was lost, and 1 returned to estrus. The results show a high in vivo developmental competence of blastocysts produced after SCNT of PDGF-APPsw-transgenic minipig fibroblasts.