Banna Mini-pig Inbred Line Research Articles

Integrated analysis of lncRNA, miRNA and mRNA expression profiles reveals regulatory pathways associated with pig testis function

Long noncoding RNA (lncRNA) and microRNA (miRNA) are known to play pivotal roles in mammalian testicular function and spermatogenesis. However, their impact on porcine male reproduction has yet to be well unraveled. Here, we sequenced and identified lncRNA and miRNA expressed in the testes of Chinese indigenous Banna mini-pig inbred line (BMI) and introduced Western Duroc (DU) and Large White (LW) pigs. By pairwise comparison (BMI vs DU, BMI vs LW, and DU vs LW), we found the gene expression differences in the testes between Chinese local pigs and introduced Western commercial breeds were more striking than those between introduced commercial breeds. Furthermore, we found 1622 co-differentially expressed genes (co-DEGs), 122 co-differentially expressed lncRNAs (co-DELs), 39 co-differentially expressed miRNAs (co-DEMs) in BMI vs introduced commercial breeds (DU and LW). Functional analysis revealed that these co-DEGs and co-DELs/co-DEMs target genes were enriched in male sexual function pathways, including MAPK, AMPK, TGF-β/Smad, Hippo, NF-kappa B, and PI3K/Akt signaling pathways. Additionally, we established 10,536 lncRNA-mRNA, 11,248 miRNA-mRNA pairs, and 62 ceRNA (lncRNA-miRNA-mRNA) networks. The ssc-miR-1343 had the most interactive factors in the ceRNA network, including 20 mRNAs and 3 lncRNAs, consisting of 56 ceRNA pairs. These factors played extremely important roles in the regulation of testis function as key nodes in the interactive regulatory network. Our results provide insight into the functional roles of lncRNAs and miRNAs in porcine testis and offer a valuable resource for understanding the differences between Chinese indigenous and introduced Western pigs.

Transcriptional Regulation of Spermatogenesis-Related Gene CDK16 in Banna Mini-Pig Inbred Line

Transcriptional Regulation of Spermatogenesis-Related Gene CDK16 in Banna Mini-Pig Inbred Line

Molecular Characteristics of SUN5 in Banna Mini-Pig Inbred Line (BMI) and Its Expression Regulation in Testis

Molecular Characteristics of SUN5 in Banna Mini-Pig Inbred Line (BMI) and Its Expression Regulation in Testis

Long- and short-read RNA sequencing from five reproductive organs of boar

The production of semen in boars involves multiple reproductive glands, including the testis (Tes), epididymis (Epi), vesicular gland (VG), prostate gland (PG), and bulbourethral gland (BG). However, previous studies on boar reproduction primarily focused on the testis, with little attention paid to the other glands. Here, we integrated single-molecule long-read sequencing with short-read sequencing to characterize the RNA landscape from five glands of Banna mini-pig inbred line (BMI) and Diannan small-ear pigs (DSE). We identified 110,996 full-length isoforms from 22,298 genes, and classified the alternative splicing (AS) events in these five glands. Transcriptome-wide variation analysis indicated that the number of single nucleotide polymorphisms (SNPs) in five tissues of BMI was significantly lower than that in the non-inbred pig, DSE, revealing the effect of inbreeding on BMI. Additionally, we performed small-RNA sequencing and identified 299 novel miRNAs across all glands. Overall, our findings provide a comprehensive overview of the RNA landscape within these five glands, paving the path for future investigations on reproductive biology and the impact of inbreeding on pig transcriptome.

Molecular characteristics and transcriptional regulatory of spermatogenesis-related gene RFX2 in adult Banna mini-pig inbred line (BMI).

RFX2 plays critical roles in mammalian spermatogenesis and cilium maturation. Here, the testes of 12-month-old adult boars of Banna mini-pig inbred line (BMI) were subjected to whole-transcriptome sequencing. The results indicated that the average expression (raw count) of RFX2 gene in BMI testes was 16138.25, and the average expression value of the corresponding transcript ENSSSCT00000043271.2 was 123.1898. The CDS of RFX2 obtained from BMI testes was 2,817 bp (GenBank accession number: OL362242). Gene structure analysis showed that RFX2 was located on chromosome 2 of the pig genome with 19 exons. Protein structure analysis indicated that RFX2 contains 728 amino acids with two conserved domains. Phylogenetic analysis revealed that RFX2 was highly conserved with evolutionary homologies among mammalian species. Other analyses, including PPI networks, KEGG, and GO, indicated that BMI RFX2 had interactions with 43 proteins involving various functions, such as in cell cycle, spermatid development, spermatid differentiation, cilium assembly, and cilium organization, etc. Correlation analysis between these proteins and the transcriptome data implied that RFX2 was significantly associated with FOXJ1, DNAH9, TMEM138, E2F7, and ATR, and particularly showed the highest correlation with ATR, demonstrating the importance of RFX2 and ART in spermatogenesis. Functional annotation implied that RFX2 was involved in 17 GO terms, including three cellular components (CC), six molecular functions (MF), and eight biological processes (BP). The analysis of miRNA-gene targeting indicated that BMI RFX2 was mainly regulated by two miRNAs, among which four lncRNAs and five lncRNAs competitively bound ssc-miR-365-5p and ssc-miR-744 with RFX2, respectively. Further, the dual-luciferase report assay indicated that the ssc-miR-365-5p and ssc-miR-744 significantly reduced luciferase activity of RFX2 3'UTR in the 293T cells, suggesting that these two miRNAs regulated the expression of RFX2. Our results revealed the important role of RFX2 in BMI spermatogenesis, making it an intriguing candidate for follow-up studies.

Transcriptomic analysis of testis and epididymis tissues from Banna mini-pig inbred line boars with single-molecule long-read sequencing†.

In mammals, testis and epididymis are critical components of the male reproductive system for androgen production, spermatogenesis, sperm transportation, as well as sperm maturation. Here, we report single-molecule real-time sequencing data from the testis and epididymis of the Banna mini-pig inbred line (BMI), a promising laboratory animal for medical research. We obtained high-quality full-length transcriptomes and identified 9879 isoforms and 8761 isoforms in the BMI testis and epididymis, respectively. Most of the isoforms we identified have novel exon structures that will greatly improve the annotation of testis- and epididymis-expressed genes in pigs. We also found that 3055 genes (over 50%) were shared between BMI testis and epididymis, indicating widespread expression profiles of genes related to reproduction. We characterized extensive alternative splicing events in BMI testis and epididymis and showed that 96 testis-expressed genes and 79 epididymis-expressed genes have more than six isoforms, revealing the complexity of alternative splicing. We accurately defined the transcribed isoforms in BMI testis and epididymis by combining Pacific Biotechnology Isoform-sequencing (PacBio Iso-Seq) and Illumina RNA Sequencing (RNA-seq) techniques. The refined annotation of some key genes governing male reproduction will facilitate further understanding of the molecular mechanisms underlying BMI male sterility. In addition, the high-confident identification of 548 and 669 long noncoding RNAs (lncRNAs) in these two tissues has established a candidate gene set for future functional investigations. Overall, our study provides new insights into the role of the testis and epididymis during BMI reproduction, paving the path for further studies on BMI male infertility.

Genome-wide single nucleotide polymorphism array and whole-genome sequencing reveal the inbreeding progression of Banna minipig inbred line.

Inbred pigs are promising animal models for biomedical research and xenotransplantation. Established in 1980, the Banna minipig inbred (BMI) line originated from a sow and its own male offspring. It was selected from a small backcountry minority Lahu village, where records show that no other pig breed has ever been introduced. During the inbreeding process, we perfomed extreme inbreeding over 23 consecutive generations using full-sibling or parent-offspring mating. In order to investigate the inbreeding effects in BMI pigs across generations over the past 40 years, in this study we conducted a genome-wide SNP genotyping of the last 10 generations, representing generations 14-23. In total, we genotyped 57,746 SNPs, corresponding to an average decrease in heterozygosity rate of 0.0078 per generation. Furthermore, we were only able to identify 18,216 polymorphic loci with a MAF larger than 0.05, which is substantially lower than the values in previous reports on other pig breeds. In addition, we sequenced the genome of the first pig in the twenty-third generation (inbreeding coefficient 99.28%) to an average coverage of 12.4× to evaluate at the genome level the impact of advanced inbreeding. ROH analysis indicates that BMI pigs have longer ROHs than Wuzhishan and Duroc pigs. Those long ROH regions in BMI pigs are enriched for distinct functions compared with the highly polymorphic regions. Our study reveals a genome-wide allele diversity loss during the progress of inbreeding in BMI pigs and characterizes ROH and polymorphic regions as a result of inbreeding. Overall, our results indicate the successful establishment of the BMI line, which paves the way for further in-depth studies.

Septin14, a gene specifically expressed in the testis and seminal vesicle of the Banna mini-pig inbred line (BMI).

Septin14 is an important spermatogenesis related gene involved in the pathogenesis of male infertility that has not been well studied. Here, full-length Septin14 cDNA of the Banna mini-pig inbred line (BMI) was cloned using the RACE method and expressed in pig kidney epithelial cells (PK15) and E. coli Rosetta (DE3) cells. Septin14 expression was identified in somatic tissues and testis in different developmental stages. The pig Septin14 CDS is 1,299 bp long, and encodes a peptide (or protein) of 432 amino acids (MW=50.4 kDa). Phylogenetic analysis indicated that pig Septin14 was highly evolutionarily conserved. Subcellular localization of GFP-tagged Septin14 fusion protein revealed that Septin14 was distributed throughout the testicular cells. Among 34 pig tissues, Septin14 mRNA was found specifically in testis and seminal vesicle. In six different postnatal developmental stages, the testicular level of Septin14 mRNA was barely detectable on day 2, while the highest level occurred on day 75. The spatiotemporal expression profile of Septin14, reported herein for the first time in pig, indicated that Septin14 might be involved in the division, development and apoptosis of germ cells. Furthermore, using a pET prokaryotic expression system, we expressed and isolated recombinant 67.9 kDa Septin14 protein.

Effect of Prokaryotic Expressed Nucleoplasmin on the Efficiency of Somatic Cell Nuclear Transfer in Banna Mini-pig Inbred Line

Effect of Prokaryotic Expressed Nucleoplasmin on the Efficiency of Somatic Cell Nuclear Transfer in Banna Mini-pig Inbred Line

Cloning, sequence characterization, and expression patterns of members of the porcine TSSK family.

Testis-specific serine kinases (TSSKs) are a family of serine/threonine kinases highly expressed in the testes that are responsible for regulating many spermatogenesis-related protein activities. Mutations in this family have a positive relationship with oligospermia and azoospermia in human and mouse. Here, five members of the TSSK family from a Banna mini-pig inbred line (BMI) were cloned, sequenced, and characterized. The full-length coding sequences of BMI TSSKs varied from 807 (TSSK3) to 1095 bp (TSSK1) and encoded 268 to 364 amino acids with molecular weights in the range 30.11 to 41.34 kDa. Following comparison with TSSK4 genes in other species, BMI TSSK4 was found to contain three alternatively spliced variants, inform1, inform 3, and inform 4. BMI TSSK1 and TSSK2 are co-localized on the Sus scrofa chromosome (SSC) 14, and consist of a single exon; TSSK3, TSSK4, and TSSK6 are on SSC6, SSC7, and SSC2, and consist of two, four, and one exon, respectively. Multiple protein sequence alignment and phylogenetic analysis showed that the regions spanning the S_TKc domains were more conserved between pig and other animals: with TSSK1 and TSSK2 and TSSK3 and TSSK6 displaying the greatest degree of homology across species, and the TSSK4 protein clearly distinct from other members. Multi-tissue RT-PCR showed BMI TSSK1, TSSK3, and TSSK4 were only expressed in the testes and seminal vesicle, TSSK2 was confined to testes only, while TSSK6 was expressed widely in adult tissues but was highest in the testes.

Overexpression of Banna mini-pig inbred line fatty acid binding protein 3 promotes adipogenesis in 3T3-L1 preadipocytes.

Fatty acid binding protein 3 (H-FABP, FABP3) has been significantly associated with intramuscular fat (IMF) content in pigs, which is positively correlated with palatability of pork. However, its underlying function is not fully elucidated. We have investigated the effects of overexpression of the FABP3 gene on differentiation and adipogenesis of 3T3-L1 preadipocytes in the fat Banna mini-pig inbred line (fBMIL). Eukaryotic vectors that expressed the FABP3 protein were constructed, and stably established in the 3T3-L1 preadipocytes cell line. Cells were induced in a standard differentiation cocktail. Morphological changes and the degree of adipogenesis were measured by Oil Red O staining assay and triacylglycerol content measurement, respectively. mRNA expression levels of triacylglycerol metabolism-related genes were measured by qPCR. FABP3 significantly promoted differentiation of 3T3-L1 cells and enhanced triacylglycerol levels (P < 0.05). mRNA of the peroxisome proliferator-activated receptor γ (PPARγ), adipocyte fatty acid binding protein (422/aP2) and glycerol-3-phosphate dehydrogenase (GPDH) gene increased markedly (P < 0.05). In conclusion, expression of the FABP3 gene enhances adipogenesis in 3T3-L1 preadipocytes primarily by upregulating lipogenic PPARγ, 422/aP2 and GPDH genes.

Highly Efficient Generation of GGTA1 Biallelic Knockout Inbred Mini-Pigs with TALENs

Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.

Complementary DNA cloning, sequence analysis, and tissue transcription profile of a novel U2AF2 gene from the Chinese Banna mini-pig inbred line

U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an important gene for pre-messenger RNA splicing in higher eukaryotes. In this study, the Banna mini-pig inbred line (BMI) U2AF2 coding sequence (CDS) was cloned, sequenced, and characterized. The U2AF2 complete CDS was amplified using the reverse transcription-polymerase chain reaction (RT-PCR) technique based on the conserved sequence information of cattle and known highly homologous swine expressed sequence tags. This novel gene was deposited into the National Center for Biotechnology Information database (Accession No. JQ839267). Sequence analysis revealed that the BMI U2AF2 coding sequence consisted of 1416 bp and encoded 471 amino acids with a molecular weight of 53.12 kDa. The protein sequence has high sequence homology with U2AF65 of 6 species - Homo sapiens (100%), Equus caballus (100%), Canis lupus (100%), Macaca mulatta (99.8%), Bos taurus (74.4%), and Mus musculus (74.4%). The phylogenetic tree analysis revealed that BMI U2AF65 has a closer genetic relationship with B. taurus U2AF65 than with U2AF65 of E. caballus, C. lupus, M. mulatta, H. sapiens, and M. musculus. RT-PCR analysis showed that BMI U2AF2 was most highly expressed in the brain; moderately expressed in the spleen, lung, muscle, and skin; and weakly expressed in the liver, kidney, and ovary. Its expression was nearly silent in the spinal cord, nerve fiber, heart, stomach, pancreas, and intestine. Three microRNA target sites were predicted in the CDS of BMI U2AF2 messenger RNA. Our results establish a foundation for further insight into this swine gene.

Molecular Cloning and Characterization of a Novel Gene STXBP6 from Chinese Banna Mini-Pig Inbred Line (BMI)

Molecular Cloning and Characterization of a Novel Gene STXBP6 from Chinese Banna Mini-Pig Inbred Line (BMI)

Molecular Cloning, Sequence Characterization and Tissue Transcription Profile of a Novel Gene PQBP1 from Chinese Banna Mini-Pig Inbred Line (BMI)

Molecular Cloning, Sequence Characterization and Tissue Transcription Profile of a Novel Gene PQBP1 from Chinese Banna Mini-Pig Inbred Line (BMI)

Identification, mRNA Expression and Characterization of a Novel CENPP Gene from Chinese Banna Mini-Pig Inbred Line (BMI)

Identification, mRNA Expression and Characterization of a Novel CENPP Gene from Chinese Banna Mini-Pig Inbred Line (BMI)

Molecular Cloning, mRNA Expression and Characterization of a Novel FAIM1 Gene from Chinese Banna Mini-Pig Inbred Line (BMI)

Molecular Cloning, mRNA Expression and Characterization of a Novel FAIM1 Gene from Chinese Banna Mini-Pig Inbred Line (BMI)

The Association of MC1R Gene with Coat Color of Banna Mini-Pig Inbred Line (BMI)

The Association of MC1R Gene with Coat Color of Banna Mini-Pig Inbred Line (BMI)

CDNA Cloning, Sequence Identification and Tissue Expression Profile of a Novel Gene FAR1 From Banna Mini-Pig Inbred Line (BMI)

cDNA Cloning, Sequence Identification and Tissue Expression Profile of a Novel Gene FAR1 From Banna Mini-Pig Inbred Line (BMI)

Establishment and biological characteristics of fetal fibroblast cell lines in Banna minipig inbred line

Useing a 47 d fetus of Banna minipig inbred line as materials,fetal fibroblast cells were dispersed in vitro with trypsinnization and then subcultured.Observations on morphology,determination of viability,drawing of dynamic growth,and analysis of karyotype were carried out.The results showed that the cell was morphologically consistent with fibroblasts,cell viability was 98.06% before freezing and 92.12% after thawing,the growth curves was sigmoidal with population doubling time(PDT)of cells was approximately 36 h,karyotyping indicated a total chromosomes number 2n=38,sex chromosomes were XY,and the karyotype is normal afrer cultured up to passage 14 in vitro.