Changes in Skeletal Muscle and Body Weight on Sleeping Beauty Transposon-Mediated Transgenic Mice Overexpressing Pig mIGF-1

Abstract

Insulin-like growth factor (IGF-I) is an important growth factor in mammals, but the functions of the local muscle-specific isoform of insulin-like growth factor 1 (mIGF-1) to skeletal muscle development have rarely been reported. To determine the effect of pig mIGF-1 on body development and muscle deposition in vivo and to investigate the molecular mechanisms, the transgenic mouse model was generated which can also provide experimental data for making transgenic pigs with pig endogenous IGF1 gene. We constructed a skeletal muscle-specific expression vector using 5'- and 3'-regulatory regions of porcine skeletal α-actin gene. The expression cassette was flanked with Sleeping Beauty transposon (SB)-inverted terminal repeats. The recombinant vector could strongly drive enhanced green fluorescence protein (EGFP) reporter gene expression specifically in mouse myoblast cells and porcine fetal fibroblast cells, but not in porcine kidney cells. The EGFP level driven by α-actin regulators was significantly stronger than that driven by cytomegalovirus promoters. These results indicated that the cloned α-actin regulators could effectively drive specific expression of foreign genes in myoblasts, and the skeletal muscle-specific expression vector mediated with SB transposon was successfully constructed. To validate the effect of pig mIGF-1 on skeletal muscle growth, transgenic mice were generated by pronuclear microinjection of SB-mediated mIGF-1 skeletal expression vector and SB transposase-expressing plasmid. The transgene-positive rates of founder mice and the next-generation F1 mice were 30% (54/180) and 90.1% (64/71), respectively. The mIGF-1 gene could be expressed in skeletal muscle specifically. The levels of mRNA and protein in transgenic mice were 15 and 3.5 times higher, respectively, than in wild-type mice. The body weights of F1 transgenic mice were significantly heavier than wild-type mice from the age of 8 weeks onwards. The paraffin-embedded sections of gastrocnemius from 16-week-old transgenic male mice showed that the numbers of myofibers per unit were increased in comparison with those in the wild-type mice. mIGF-1 overexpression in mice skeletal muscle may promote myofibers hypertrophy and muscle production, and increased the average body weight of adult mice. Transgenic mice models can be generated by the mediation of SB transposon with high transgene efficiency.

Keywords: Pig insulin-like growth factor 1; Skeletal α-actin gene regulator; Sleeping Beauty transposon; Transgenic mice.

Fig. 1

Schematic diagram of skeletal-specific expression vector mediated by Sleeping Beauty (SB) transposon. ITR: inverted terminal repeat of SB; α-actin 5 promoter: 5′-regulator for skeletal α-actin gene; mIGF-1: mIGF-1 open reading frame; α-actin 3 UTR: 3′-untranslated region and contiguous 3′-noncoding region for skeletal α-actin gene; En: SV40 enhancer

Fig. 2

Green fluorescent protein expression in different cell types. The vector pT2/A-actin-EGFP was transfected into a mouse myoblast (C2C12), b porcine fetal fibroblast (PEF), and c porcine kidney (PK15) cells. pEGFP-N1 was used as a positive control

Fig. 3

F₀ founder mice were tested for transgene integration. a Mouse tail genomic DNA was subjected to PCR using three pairs of specific primers designed according to transgene sequences: α-actin-5′ (206 bp), α-actin-3′ (251 bp), pIGF-1-flank (545 bp). 1–20, F₀ founders; M, 100 bp DNA ladder; -, Wt mouse; b PCR-positive individuals were tested by Southern blot analysis. Mouse tail genomic DNA was digested with EcoRV and hybridized with DIG-labeled probes. +, pT2/A-actin-IGF-1/EcoRV; M, DNA molecular weight marker III labeled with Digoxigenin; 1–9, PCR-positive F₀ founders, -, Wt mouse

Fig. 4

IGF-1 gene expression in transgenic mouse tissues. a IGF-1 mRNA expression levels determined by real-time quantitative PCR; b IGF-1 protein levels determined by Western blot analysis

Fig. 5

IGF-1 levels of gastrocnemius from Wt and pmIGF-1 Tg mice by Western blotting

Fig. 6

Growth curves of transgenic mice. Tg transgenic mice, Wt wild-type mice. *P < 0.05 (determined using the Student’s t test, comparing Wt and Tg mice)

Fig. 7

Hematoxylin and eosin histology of 16-week-old Tg and Wt mouse gastrocnemius. Images revealed the fiber hypertrophy in pmIGF-1 Tg. The magnification was ×100 and ×400 (inset)