Cloning and functional verification of a porcine adipose tissue-specific promoter.

BACKGROUND: Fat deposition is an important economic trait in pigs. In the past decades, many genes regulating porcine fat deposition were identified by Omics technology and verified by cell biology studies. Using genetically modified pigs to investigate the function of these genes in vivo is necessary before applying in breeding. However, lack of tissue-specific promoters of pigs hinders the generation of adipose tissue-specific genetically modified pigs. RESULTS: In order to identify a porcine adipose tissue-specific promoter, we used the software Digital Differential Display (DDD) to screen 99 genes highly expressed in porcine adipose tissue. GO and KEGG enrichment analysis indicated that the 99 genes were mainly related to lipid metabolism. Q-PCR proved that LGALS12 was an adipose tissue-specific gene. Five truncated fragments of the LGALS12 promoter were cloned and the 4 kb fragment (L-4 kb) exhibited a high level of promoter activity in adipocytes and no promoter activity in non-adipocytes. Following co-transfection with adipogenic transcription factors, the promoter activity of L-4 kb was enhanced by PPARγ, C/EBPß, and KLF15, whereas it was suppressed by KLF4. Finally, we demonstrated that L-4 kb can drive APOR gene expression to exert its function in adipocytes. CONCLUSIONS: This study demonstrates that porcine LGALS12 is an adipose tissue-specific gene, and identified the 4 kb fragment of LGALS12 promoter that exhibited adipocyte-specific promoter activity. These results provide new evidence for understanding porcine fat deposition and a promoter element for adipose tissue-specific genetic modification in pigs. HIGHLIGHTS: Identified porcine LGALS12 as an adipose tissue-specific gene. Truncated LGALS12 promoter (L-4 kb) showed adipose tissue-specific promoter activity. Identified transcription factors involved in the regulation of L-4 kb promoter activity.

Adipose-Tissue-Specific Expression of Pig ApoR Protects Mice from Diet-Induced Obesity.

Fat deposition is one of the most important economic traits of pigs. Decreasing the subcutaneous fat and increasing the intramuscular fat are believed to be an effective way to improve pork quality, which is one of the main goals of pig breeding. Identifying key genes that control porcine lipid metabolism is essential for achieving this goal. Apolipoprotein R (apoR) was identified as the crucial molecule in the process of pig adipose reduction by clenbuterol. In this study, transgenic mice with adipose-tissue-specific overexpression of pig apoR (apoR mice) were constructed. The apoR mice gained less weight than wild-type (WT) mice after 18 weeks of feeding a high-fat diet. A comparison of organs between the two genotypes revealed that the weight of white adipose tissue, including inguinal and epididymal fat tissue, was significantly decreased and the weight of liver tissue was increased in apoR mice compared with WT mice. Glucose and insulin intolerance tests showed that the glucose metabolism of apoR mice was similar to that of WT mice. Histological staining proved that the adipocytes of apoR mice had a reduced average size, and gene expression analysis indicated that lipolysis in the adipose tissue of apoR mice was enhanced. Finally, the primary culture of inguinal adipocytes revealed that apoR promotes lipolysis via the Erk1/2 pathway. Taken together, the results indicate that adipose-tissue-specific expression of pig apoR protects mice from diet-induced obesity by enhancing lipolysis.

Deletion of CD163 Exon 7 Confers Resistance to Highly Pathogenic Porcine Reproductive and Respiratory Viruses on Pigs.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is a severe infectious disease in the swine industry. PRRSV infection is mediated by porcine CD163 (pCD163). Scavenger receptor cysteine-rich domain 5 coded by exon 7 of pCD163 is essential for PRRSV infection. In this study, we generated CD163 exon 7 deleted (CD163E7D) pigs using CRISPR/Cas9 mediated homologous recombination and somatic cell nuclear transfer (SCNT). The deletion of exon 7 had no adverse effects on CD163-associated functions. Pigs were further challenged with a highly pathogenic PRRSV (HP-PRRSV) strain. The CD163E7D pigs exhibited mild clinical symptoms and had decreased viral loads in blood. All CD163E7D pigs survived the viral challenge, while all the WT pigs displayed severe symptoms, and 2 out of 6 WT pigs died during the challenge. Our results demonstrated that CD163 exon 7 deletion confers resistance to HP-PRRSV infection without impairing the biological functions of CD163.

Generation of an MC3R knock-out pig by CRSPR/Cas9 combined with somatic cell nuclear transfer (SCNT) technology.

BACKGROUND: Melanocortin 3 receptor (MC3R), a rhodopsin-like G protein-coupled receptor, is an important regulator of metabolism. Although MC3R knock-out (KO) mice and rats were generated in earlier studies, the function of MC3R remains elusive. Since pig models have many advantages over rodents in metabolism research, we generated an MC3R-KO pig using a CRSPR/Cas9-based system combined with somatic cell nuclear transfer (SCNT) technology. METHOD: Four CRSPR/Cas9 target vectors were constructed and then their cleavage efficiency was tested in porcine fetal fibroblasts (PFFs). The pX330-sgRNA1 and pX330-sgRNA4 vectors were used to co-transfect PFFs to obtain positive colonies. PCR screening and sequencing were conducted to identify the genotype of the colonies. The biallelically modified colonies and wild-type control colonies were used simultaneously as donor cells for SCNT. A total of 1203 reconstructed embryos were transferred into 6 surrogates, of which one became pregnant. The genotypes of the resulting piglets were determined by PCR and sequencing, and off-target effects in the MC3R KO piglets were detected by sequencing. Then, offspring were obtained through breeding and six male KO pigs were used for the growth performance analysis. RESULTS: Four vectors were constructed successfully, and their cleavage efficiencies were 27.96, 44.89, 32.72 and 38.86%, respectively. A total of 21 mutant colonies, including 11 MC3R-/- and 10 MC3R+/- clones, were obtained, corresponding to a gene targeting efficiency of 29.17%, with 15.28% biallelic mutations. A total of 6 piglets were born, and only two MC3R KO piglets were generated, one with malformations and a healthy one. No off-target effects were detected by sequencing in the healthy mutant. Six male MC3R KO pigs were obtained in the F2 generation and their body weight and body fat were both increased compared to wild-type full siblings. CONCLUSION: A MC3R KO pig strain was generated using the CRSIPR/Cas9-based system, which makes it possible to study the biological function of MC3R in a non-rodent model.

[Molecular cloning, expression profile analysis and construction of adipose tissue specific expression vector of pig Gli1 gene].

The Hedgehog (Hh) signaling pathway inhibits fat accumulation, which is conserved in a wide variety of organisms from Drosophila to vertebrates, but few reports about its effect on pigs are available. In this study, pig Gli1 gene was cloned for the first time by rapid amplification of cDNA ends (RACE) and RT-PCR. Pig Gli1 expression profiles were then studied in different tissues and in different developmental stages of the adipose tissue of pigs using real-time PCR. Finally, the eukaryotic expression vector and the adipose tissue specific expression vector were constructed. The results showed that the full pig Gli1 cDNA length was 3 576 bp, the genomic sequence contained 10 715 bp with 12 exons, and 1 106 amino acids were encoded. Pig Gli1 was predicted as an unstable hydrophilic protein without a tans-membrane structure or a signal peptide. The C2H2 zinc finger domain and a nuclear localization sequence were found in pig Gli1. A homology analysis of the Gli1 amino acids and the genomic sequences among seven species showed that the identities were all greater than 80%, which indicates that Gli1 is highly conserved among different species. Tissue expression profile analysis showed that pig Gli1 was only expressed in the tone tissue of adult pigs. Analysis of the pig adipose tissue developmental process showed that Gli1 was detected in the adipose tissue of one-week-old pigs, but not in one-month-old and three-month-old pigs. Finally, a pig Gli1 eukaryotic expression vector was constructed and properly expressed with cell transfection. An adipose tissue specific expression vector was constructed for transgenic animal studies.