Fig 1.
Structure of pTet retroviral multi-cistronic vector containing hAPP, hTau, and hPS1 under the control of the CMVE and hPDGFβ promoter.
(A) Linear map. (B) Circular map. (C) Protein expression of AD transgenes in HEK cells. CMVE, human CMV enhancer; hPDGFβ, human platelet–derived growth factor β; hAPP, human amyloid precursor protein; hTau, human tau protein; hPS1, human presenilin 1. Three different gene product were detected by Western blot analysis with antibody against hAPP (22C11), Tau (Tau-5), PS1 (PS1-CTF). S.M: protein size marker, Mock and Multi; control cell and multi-cistronic vector transfected cell, repectively.
Table 1.
Oligonucleotides for cloning and mutagenesis.
Fig 2.
Preparation of AD transgenic Jeju black pig ear fibroblasts (JB-PEFAD).
Morphology (A), integration (B), and mRNA expression (C) of AD multi-cistronic vector in JB-PEFAD cells. Karyotype (D) of JB-PEFAD cell lines, revealing normal chromosome number (2n = 38). AD multi-cistronic vector: hAPP, human amyloid precursor protein; hTau, human tau protein; PS1, presenilin 1. Scale bars = 20 μm.
Fig 3.
Morphology (A) of blastocysts derived from SCNTAD embryos on day 7, and developmental rate (B) of SCNTAD embryos. The gDNAintegrationrates (C) of AD transgenes in individual blastocysts, determined by PCR. Expression levels (D) in five blastocysts derived from SCNT embryos using transgenic cell line #2-1.hAPP, human amyloid precursor protein; hTau, human tau protein; PS1, presenilin 1. Scale bars = 120 μm.
Fig 4.
Production of AD transgenic cloned pigs and identification of AD transgenes (A), hAPP, hTau, and hPS1 in cloned pigs: #1, 2 (alive, B), and 3 (stillborn). Expression of APP-Tau and Tau-PS1 in the tissues of AD transgenic pig, determined by real-time RT-PCR (C) and expression of AD-related genes in brain of TG pigs (D). hAPP, human amyloid precursor protein; hTau, human tau protein; hPS1: human presenilin 1. The experiment was repeated three or four independent times.
Table 2.
Production of AD transgenic piglets.
Table 3.
Full-term development of AD transgenic piglets.
Table 4.
Microsatellite (MS) analysis of donor surrogate, donor cell, and offspring.
Fig 5.
Bio-marker analysis of Tg pigs.
(A) Quantification of Aβ1–40, Aβ1–42, and total Tau in the cortex of control (blank box) and Tg brains (black bar) using ELISA. All three gene expression was elevated in the TG brain. (B) Expression of APP in the cortex region of JNUPIGs confirmed by immunohistochemistry stained with anti-Aβ specific antibody. (C) Astrocyte activation, monitored by GFAP level, in control and Tg brain. GFAP level was highly elevated in TG brain. Images, taken at 400× magnification, are representative of at least four sections per animal. Data were analyzed by independent samples t-test in SPSS (*: p < 0.05, **: p < 0.01). The experiment was repeated three independent times.
Fig 6.
Predicted multiple insertion sites in chromosome X of transgenic pigs.
The insertion site was determined by NGS analysis of gDNA of a transgenic pig. The transgene was inserted into the region between exons 9 and 10 of BEND2.
Fig 7.
Estimation of copy number in the transgenic piglet genome.
Real-time PCR was used to estimate the copy numbers of inserted transgenes. The X-axis indicates the inserted regions in the construct. An endogenous single-copy gene, glucagon (GCG), was used as a normalization control. Y-axis indicates 2−ΔΔCT values. The results reveal that more than five copies of the inserts were present in the genome of the transgenic pig.
Table 5.
Primers used for PCR.