Chuanying Pan

SNL fibroblast feeder layers support derivation and maintenance of human induced pluripotent stem cells

Induced pluripotent stem (iPS) cells can be derived from human somatic cells by cellular reprogramming. This technology provides a potential source of non-controversial therapeutic cells for tissue repair, drug discovery, and opportunities for studying the molecular basis of human disease. Normally, mouse embryonic fibroblasts (MEFs) are used as feeder layers in the initial derivation of iPS lines. The purpose of this study was to determine whether SNL fibroblasts can be used to support the growth of human iPS cells reprogrammed from somatic cells using lentiviral expressed reprogramming factors. In our study, iPS cells expressed common pluripotency markers, displayed human embryonic stem cells (hESCs) morphology and unmethylated promoters of NANOG and OCT4. These data demonstrate that SNL feeder cells can support the derivation and maintenance of human iPS cells.

A DdeI PCR-RFLP detecting genetic variation of goat POU1F1 gene

We described a DdeI PCR-RFLP method for detecting silent allele at goat POU1F1 locus: p.S241S. Frequencies of D1 allele varied from 0.600 to 1.000 in eight Chinese native breeds. Association of DdeI RFLP genotypes with milk yield of dairy goat was significant (P < 0.05). Key words: Goat, POU1F1 gene, polymorphism, association, milk yield

Novel Nucleotide Variations, Haplotypes Structure and Associations with Growth Related Traits of Goat AT Motif-Binding Factor (ATBF1) Gene

The AT motif-binding factor (ATBF1) not only interacts with protein inhibitor of activated signal transducer and activator of transcription 3 (STAT3) (PIAS3) to suppress STAT3 signaling regulating embryo early development and cell differentiation, but is required for early activation of the pituitary specific transcription factor 1 (Pit1) gene (also known as POU1F1) critically affecting mammalian growth and development. The goal of this study was to detect novel nucleotide variations and haplotypes structure of the ATBF1 gene, as well as to test their associations with growth-related traits in goats. Herein, a total of seven novel single nucleotide polymorphisms (SNPs) (SNP 1-7) within this gene were found in two well-known Chinese native goat breeds. Haplotypes structure analysis demonstrated that there were four haplotypes in Hainan black goat while seventeen haplotypes in Xinong Saanen dairy goat, and both breeds only shared one haplotype (hap1). Association testing revealed that the SNP2, SNP5, SNP6, and SNP7 loci were also found to significantly associate with growth-related traits in goats, respectively. Moreover, one diplotype in Xinong Saanen dairy goats significantly linked to growth related traits. These preliminary findings not only would extend the spectrum of genetic variations of the goat ATBF1 gene, but also would contribute to implementing marker-assisted selection in genetics and breeding in goats.

Exploring the novel genetic variant of PITX1 gene and its effect on milk performance in dairy goats

Abstract Paired-like homeodomain transcription factor 1 (PITX1) plays an important role in pituitary development by indirectly regulating the expression of the GH and PRL genes, and therefore PITX1 gene is regarded as a potential candidate gene for building the relationship between the gene polymorphism and milk traits. The aim of this study was to explore the novel genetic variant in PITX1 gene and its effect on milk performance in dairy goats. Herein, a novel genetic variation (NW_00314033: g.201G>A or IVS1+41G>A) located at nt41 position of the first intron of the goat PITX1 gene was reported at the P1 locus, which can be genotyped by the Msp I PCR-RFLP. In the Msp I PCR-RFLP analyis, the GG variant was a major genotype, and the A variant was a minor allele in Guanzhong dairy goats which was at Hardy-Weinberg disequilibrium (chi-square χ2=140, P

Insertion/Deletion Within the KDM6A Gene Is Significantly Associated With Litter Size in Goat

A previous whole-genome association analysis identified lysine demethylase 6A (KDM6A), which encodes a type of histone demethylase, as a candidate gene associated to goat fecundity. KDM6A gene knockout mouse disrupts gametophyte development, suggesting that it has a critical role in reproduction. In this study, goat KDM6A mRNA expression profiles were determined, insertion/deletion (indel) variants in the gene identified, indel variants effect on KDM6A gene expression assessed, and their association with first-born litter size analyzed in 2326 healthy female Shaanbei white cashmere goats. KDM6A mRNA was expressed in all tissues tested (heart, liver, spleen, lung, kidney, muscle, brain, skin and testis); the expression levels in testes at different developmental stages [1-week-old (wk), 2, 3 wk, 1-month-old (mo), 1.5 and 2 mo] indicated a potential association with the mitosis-to-meiosis transition, implying that KDM6A may have an essential role in goat fertility. Meanwhile, two novel intronic indels of 16 bp and 5 bp were identified. Statistical analysis revealed that only the 16 bp indel was associated with first-born litter size (P < 0.01), and the average first-born litter size of individuals with an insertion/insertion genotype higher than that of those with the deletion/deletion genotype (P < 0.05). There was also a significant difference in genotype distributions of the 16 bp indel between mothers of single-lamb and multi-lamb litters in the studied goat population (P = 0.001). Consistently, the 16 bp indel also had a significant effect on KDM6A gene expression. Additionally, there was no significant linkage disequilibrium (LD) between these two indel loci, consistent with the association analysis results. Together, these findings suggest that the 16 bp indel in KDM6A may be useful for marker-assisted selection (MAS) of goats.

Pig KDM5B: mRNA expression profiles of different tissues and testicular cells and association analyses with testicular morphology traits

Lysine specific demethylase 5B gene (KDM5B, also known as JARID1B or PLU-1), encoding an enzyme of the lysine-specific histone demethylase family, has been reported to regulate androgen receptor transcriptional activity and male reproduction. To fully study the expression characteristics and genetic effects of pig KDM5B gene, the objective of this study was to investigate the mRNA expression profiles of KDM5B among different tissues and testicular cells (spermatogonia stem cells, SSCs; sertoli cells, SCs; leydig cells, LCs), as well as to explore the insertion/deletion (indel) variations of this gene. Expression analysis results revealed that the KDM5B gene was highly expressed in testis than other tissues in 7-day-old piglet (P < 0.01). In particular, this gene was highest expressed in testis of adult boar (P < 0.01). Furthermore, the KDM5B mRNA expression level in SSCs was significantly higher than those in LCs and SCs (P < 0.01). Besides, a 35-bp indel was first verified in the intron 11 of pig KDM5B gene, and the animals with insertion/insertion genotype exhibited superior testicular weight and testicular short perimeter than those with other genotypes (P < 0.05) in 40-day-old Landrace pigs. Together, these findings suggest that KDM5B plays a crucial role in male fertility and the 35-bp indel can be used for marker-assisted selection of boar.

The muscle development transcriptome landscape of ovariectomized goat

In practical production, almost all rams and about 50% of ewes are used to fatten. Researchers have proved that ewe ovariectomy could improve the productivity significantly, but the specific molecular mechanism is still unknown. In this study, five independent cDNA libraries (three and two from ovariectomized and normal ewe longissimus dorsi samples, respectively) were constructed to thoroughly explore the global transcriptome, further to reveal how the ovariectomized ewes influence muscle development by Illumina2000 sequencing technology. As a result, 205 358 transcripts and 118 264 unigenes were generated. 15 490 simple sequence repeats (SSRs) were revealed and divided into six types, and the short repeat sequence SSR (monomers, dimers, trimers) was the domain type. Single nucleotide polymorphism analysis found that the number of transition was greater than the number of transversion among the five libraries. Furthermore, 1612 differently expressed genes (DEGs) (Log2fold_change > 1 and p < 0.05) were revealed between ovariectomized and normal ewe groups, in which 903 genes were expressed commonly in the two groups, and 288 and 421 genes were uniquely expressed in normal and ovariectomized ewe groups, respectively. Gene Ontology (GO) analysis categorized all unigenes into 555 GO terms and 56 DEGs were significantly categorized into 43 GO terms (p < 0.05). KEGG enrichment analysis annotated 12 976 genes (containing 137 DEGs) to 86 pathways, among them 24 and 11 DEGs involved in development and reproduction associated pathways, respectively. To validate the reliability of the RNA-seq analysis, 22 candidate DEGs were randomly selected to perform quantitative real-time polymerase chain reaction. The result showed that 9 and 1 genes were significantly and approximately significantly expressed in control and treatment group, respectively, and the results of RNA-seq are believable in this study. Overall, these results were helpful for elucidating the molecular mechanism of muscle development of ovariectomized animals and the application of female ovariectomy in fattening.

Identification of novel alternative splicing transcript and expression analysis of bovine TMEM95 gene.

Transmembrane protein 95 (TMEM95) is closely related to male reproductive performance in cattle, but does not affect semen quality. Alternative splicing plays an important role in regulating biological function as well as in generating proteomic and functional diversity in metazoan organisms. Thus, the aim of this study was to clone and identify transcripts of the TMEM95 gene in cattle using RT-PCR, characterize them via bioinformatics analysis, and detect their expression patterns using qRT-PCR. Two transcripts of TMEM95 were identified in cattle, including TMEM95-SV1 and TMEM95-SV2. Bioinformatics predicted that TMEM95-SV1 has a leucine-rich repeat C-terminal domain and a Pfam: IZUMO. These regions are closely related to protein interactions and the acrosome reaction, respectively. Interestingly, the two transcripts were exclusively expressed in the testes and brain in male fetus cattle, and TMEM95-SV1 was expressed in the brain at significantly higher levels than in the testis (P<0.05, 4.06-fold) and TMEM95-SV2 in the brain (P<0.05, 4.95-fold). These findings enrich the understanding of the TMEM95 gene function and benefit for enhancing male reproduction in cattle industry.

Three novel single-nucleotide polymorphisms of the bovine LHX3 gene

The LHX3 gene encodes LIM homeodomain class transcription factors that have important roles to play in pituitary and nervous system development. On the one hand, mutations of LHX3 are associated with deficiencies of growth hormone (GH), prolactin (PRL), luteotrophic hormone (LH), follicle-stimulating hormone (FSH) and thyroidstimulating hormone (TSH); on the other hand, mutations of LHX3 are also associated with combined pituitary hormone deficiency (CPHD) diseases in human and animal models. To date, few polymorphisms of the bovine LHX3 gene have been reported. In this study, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods were employed to screen the genetic variations within the bovine LHX3 gene in 802 Chinese indigenous cattle. The results revealed three novel single-nucleotide polymorphisms (SNPs): AY923832: g.7553G>A, 7631C>T and 7668C>G. Among them, a synonymous mutation of exon II was identified: GAG (Glu) >GAA (Glu) at position 72 aa (AY923832:g.7553G>A) of LHX3 (403aa) in the four Chinese bovine breeds. Significant statistical differences in genotypic frequencies for exon II and its flanking region of the LHX3 gene implied that the polymorphic locus was significantly associated with cattle breeds by the χ2-test (χ2 = 68.975, df = 6, P<0.001). Hence, the three novel SNPs not only extend the spectrum of genetic variations of the bovine LHX3 gene, but could also possibly contribute to conducting association analysis and evaluating these as genetic markers in bovine breeding and genetics, and CPHD detection.

Novel genetic variants of sine oculis homeobox homolog 3 gene are associated with body weight and average daily gain in Bos taurus

Sine oculis homeobox homolog 3 (Six3) gene is responsible for normal mammalian pituitary development, and its genetic variations or deficiency will cause hypopituitarism, suggesting that this gene is a potential candidate gene for studying association with growth traits in animals. Herein, genetic variants within bovine Six3 gene was screened in 1031 individuals from four Chinese indigenous cattle breeds. Two novel polymorphisms (NC_007309:g.2515G>A and NC_007309:g.2607T>C) locating at positions nt1707 and nt1799 of intron 1 in bovine Six3 gene, were found, and could be genotyped by TaqI ACRS PCR-RFLP and Alw26I PCR-RFLP, respectively. The frequencies of allele “A” of TaqI locus varied from 0.004 to 0.309, as well as the frequencies of allele “C” of Alw26I locus waved from 0.025 to 0.340. Association analysis revealed no significant association of TaqI locus with growth traits in Nanyang breed. However, significant relationships between Alw26I locus and body weight (BW) and average daily gain (ADG) in Nanyang breed was found (p<0.05). The individuals with genotype TC had greater body weight and average daily gain than those with genotype TT at 18 months old. Furthermore, based on combinated genotypes from these two loci, diplotypes was found to be associated with growth traits (p<0.05).The individuals with dihaplotype GG-TC had greater body weight and average daily gain at 18 month-old than those of other dihaplotypes. Therefore, the TaqI and Alw26I genetic variants of bovine Six3 gene were recommended as DNA markers related to growth traits through marker-assisted selection for genetics and breeding in cattle.