世界首例可发红黄绿青四种荧光的克隆猪（附原文）

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Abstract
# |' X- x; k, b0 @' ~1 JMultiple genetic modifications in pigs can essentially benefit research on agriculture, human disease and xenotransplantation.9 N* }. e8 X, c  d+ I) r; k
Most multi-transgenic pigs have been produced by complex and time-consuming breeding programs using multiple
7 K6 u9 [. J6 n! D3 d" K- B0 Psingle-transgenic pigs. This study explored the feasibility of producing multi-transgenic pigs using the viral 2A peptide in
2 }, q* l- k& }+ D3 v* a6 A% Fthe light of previous research indicating that it can be utilized for multi-gene transfer in gene therapy and somatic cell$ }9 Z! V  L# \% G) o2 Z& `
reprogramming. A 2A peptide-based double-promoter expression vector that mediated the expression of four fluorescent
: c$ x/ W' I. X1 V: q! _+ S& Qproteins was constructed and transfected into primary porcine fetal fibroblasts. Cell colonies (54.3%) formed under G418
6 o' ?& C# ^' D1 `* ~. k1 Yselection co-expressed the four fluorescent proteins at uniformly high levels. The reconstructed embryos, which were* g0 d5 ?+ b( o
obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted/ u- s$ e/ F3 u) y( l
into seven recipient gilts. Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent
( k- k& J& f1 [* v2 kproteins at equivalently high levels in various tissues. The fluorescence intensities were directly observed at the nose, hoof, J& K; b: f% P4 S" @
and tongue using goggles. The results suggest that the strategy of combining the 2A peptide and double promoters) _8 w: U! S- z+ U
efficiently mediates the co-expression of the four fluorescent proteins in pigs and is hence a promising methodology to
# l) \6 I4 J* P& r1 Wgenerate multi-transgenic pigs by a single nuclear transfer.