[show abstract][hide abstract] ABSTRACT: Desmoglein 4 (DSG4) plays an important role in the regulation of growth and differentiation of hair follicles in mammals. In this study, a 755 bp long segment of DSG4 was screened in 544 sheep sampled from nine Chinese indigenous breeds and two Western breeds using PCR-SSCP assay with three different pairs of primers. Two of the three fragments showed polymorphisms with genotypes defined as AA, AB, BB and BC, and DD, DE, and EE, respectively. Interestingly, polymorphisms in these two fragments were in strong linkage disequilibrium. Only three haplotypes were found, of which haplotype AD determined by alleles A and D was the major one in all breeds, while haplotype BE was only found in Chinese breeds that possess divergent frequencies ranging from 0.02 to 0.43; haplotype CD was very rare and present in only one Chinese sheep. Sequences of the three haplotypes showed seven single nucleotide polymorphisms (SNPs) and a TTG insertion/deletion (indel), leading to five amino acid substitutions and a glycine indel. Our study provides valuable genetic markers in evaluating the impact of the DSG4 gene on wool traits in sheep.
AFRICAN JOURNAL OF BIOTECHNOLOGY 08/2011; 10:6852-6856. · 0.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: The glial cell-derived neurotrophic factor (GDNF) is a member of the transforming growth factor beta (Tgf-beta) superfamily, which is produced by Sertoli cells and plays an important role in the proliferation and differentiation of spermatogonial stem cells (SSC). The addition of proper amount of GDNF to the culture media can promote SSC proliferation in vitro. Besides, GDNF regulates the self-renewal and differentiation of SSCs through various signaling pathways. This review focuses on the effects of GDNF on the proliferation and differentiation of mammalian SSCs and GDNF-mediated signaling pathways.
Zhonghua nan ke xue = National journal of andrology 07/2011; 17(7):628-33.
[show abstract][hide abstract] ABSTRACT: The Mongolian sheep ear marginal tissue fibroblast cell line (MSF32) from 32 samples was successfully established by using primary explants technique and cell cryoconservation technology. MSF32 cells were adherent, with a population doubling time of 28.2 h. Chromosome analysis showed that >90.2% of cells were diploid (2n=54) prior to cell passage 4. Isoenzyme analyses of lactate dehydrogenase and malate dehydrogenase showed that the MSF22 cells had no cross-contamination with other species. Tests for cell line contamination with bacteria, fungi, viruses and mycoplasmas were also negative. Plasmids encoding the fluorescent proteins pEGFP-N3, pEGFP-C1, pECFP-N1, pECFP-mito, pDsRed1-N1 and pEYFP-N1 were transfected into cells to study exogenous gene expression in the cells. The plasmid transfection efficiency was between 12.3% and 63.3%. Every index of the MSF32 cell line meets all the standard quality controls of American Type Culture Collection (ATCC). Not only has the genetic resources of the Mongolian sheep been preserved at the cell level, but also valuable materials had been provided for genome, postgenome and somacloning research.
[show abstract][hide abstract] ABSTRACT: Although the avian primordial germ cells (PGCs) have been used to produce transgenic birds, their characteristics largely remain unknown. The isolation, culture, biological characterization, and directed neural differentiation of duck EG cells were assayed in this study. The Results showed that the EG cells were got by isolating embryonic gonad and surrounding tissue from 7-day-old duck embryo. The PGCs co-cultured with their gonadal somatic cells were well grown. After passaging, the EG cells were incubated in medium with cytokines and Mitomycin C on inactivated duck embryonic fibroblasts (DEFs) feeder layers. After several passages, alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) resulted positive, cellular markers detection positive for SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. Karyotype analysis showed the EG cells kept diploid condition and the hereditary feature was stable in accordance with varietal characteristics of duck. These cells grew continuously for 11 passages on DEFs. Under induction of medium with BME, RA, and IBMX, the EG cells lost undifferentiated state, large amount of neural cells appeared with the formation of neural cells networks. Special Nissl body was found by toluidine blue stain after induced for 7 days. Immunofluorescence staining results indicated that differentiated EG cells expressed Nestin, NSE, and GFAP positive. The expression of Nestin, NSE, and GFAP mRNA were positive by RT-PCR. The results revealed that RA can obviously promote the directed differentiation of duck EG cells into neural lineage. The duck EG cells will be useful for the production of transgenic birds, for cell replacement therapy and for studies of germ cell differentiation.
Journal of Cellular Biochemistry 02/2011; 112(6):1514-23. · 3.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×10(6) pfu/ml and 1.62×10(9) pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142 bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers.
International journal of biological sciences 01/2010; 6(6):584-9. · 3.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: After Human Gene Project, studying the kinetics of DNA translocation through a nanopore and developing a novel fast DNA sequencing technology via nanopore have become the hot spot in genetic research. We reviewed recent progress in the researches of DNA molecules analyzed with nanopores.
[show abstract][hide abstract] ABSTRACT: In the Hanasi scenic spot of the Altai Region, Xinjiang Uygur Autonomous Region, China, there is a special population known as Xinjiang Tuvinians for short. These Tuvinians were classified as Mongolians in the early 1950s by the National Ethnic Affairs Commission of China, but they claimed that they have an independent origin. To resolve this dispute and their genetic relationships with the people in the neighboring regions, we randomly selected 150 male Tuvinians in the Altai Region. Fourteen Y chromosomal markers were genotyped and eleven haplogroups were constructed. The frequencies of the haplogroups K-M9 and Q-M242 were higher in Xinjiang Tuvinians or Tuvinians in the Tuva Republic than those in the other populations (e.g., Mongolians and Kazakh). Principal component analysis , multi-dimensional scaling analysis and further phylogenetic tree analysis revealed that the Xinjiang Tuvinians were far separated from Mongolians and Kazakh. Based on these results, we proposed that Xinjiang Tuvinians are genetically distinct from Mongolians and Kazakh.
[show abstract][hide abstract] ABSTRACT: PCR-SSCP and DNA sequencing approaches were applied to assess the single nucleotide polymorphisms (SNPs) and analyze the genetic polymorphisms at partial exon 2 and intron 2 of H-FABP(Heart fatty acid-binding protein)gene, and five sheep populations that comprised of Small-Tailed Han sheep (SH, 48), Ningxia Tan sheep (Tan, 121), Tan x SH F1 (23), Poll Dorset (48) and Suffolk (24) sheep were screened in this study. The result showed: (1)four SNPs at 981(G/A), 1014(A/C) 1019(T/C) and 1058 (-/G ) and nine genotypes (AA, BB, CC, AB, AC, BC, AD, CD and BD) were detected using primer 2, the AA was the predominant genotype. A chi-square analysis suggested that the allele frequencies and genotype frequencies of H-FABP were in Hardy-Weinberg equilibrium except the Tan and Suffolk populations. Statistical analysis revealed a low polymorphism information content (PIC) in the Suffolk and Tan x SH F1 populations (PIC amp; 0.25) but an intermediate PIC in the remaining three populations (0.25 amp; PIC amp;0.50). It meant that the fragment of H-FABP had polymorphisms, which could be used as a candidate gene associated gene with phenotypic traits like intramuscular fat content in different sheep populations. (2)Three genotypes (HH, Hh and hh) determined by a SNP at 2407(T-C) were detected using primer 4. The genotype frequencies were in the order of HHHhhh. A chi-square analysis suggested that the allele frequencies and genotype frequencies of H-FABP were in Hardy-Weinberg equilibrium in the Tan and Poll Dorset populations, and the PIC values were low (PIC amp; 0.25). However, there was no polymorphisms in SH, Tan x SH F1 and Suffolk populations.
[show abstract][hide abstract] ABSTRACT: As the fast pace of genomic research continues to identify mitochondrial lineages in animals, it has become apparent that many independent studies are needed to support a robust phylogenetic inference. The aim of this study was thus to further characterize the maternal lineage, proposed to originate in southwestern region of China, using a wider survey of diverse goat breeds in China. To this end, we sequenced the mitochondrial hypervariable region 1 (HVR1) of the mtDNA control region in 145 goats of 12 Chinese breeds. Phylogenetic analysis revealed that Chinese goats were classified into four distinct lineages (A, B, C and D) as previously reported. A Mantel test and the analysis of Analysis of Molecular Variance (ANOVA) indicated that there was not an obvious geographic structure among Chinese goat breeds. Population expansion analysis based on mismatch distribution and Fu's Fs statistic indicate that two expansion events in Chinese goats occurred respectively at about 11 and 29 mutational time units ago, revealing two star-like subclades in lineage B corresponding to two population expansion events. Moreover, lineage B sequences were presented only in the breeds of southwestern or surrounding regions of China. Multiple lines of evidence from this study and previous studies indicate that for Chinese goats mtDNA lineage B originated from the southwestern region of China.
[show abstract][hide abstract] ABSTRACT: The specific expression of alpha1-AGP gene in eight different tissues of Beijing fatty chicken was investigated by RT-PCR. The full-length cDNA of alpha1-AGP was inserted into pEGFP-C1 multi-cloning sites to construct recombinant eukaryotic expression vector pEGFP-alpha1-AGP. The lipofectin method was used to transfect the pEGFP-alpha1-AGP into Beijing fatty chicken fibroblast cells. The open reading frame of Beijing fatty chicken alpha1-AGP gene was 612 base pairs in length, which was expressed higher in liver and lung than in muscle. This gene did not express in heart and kidney. The expression efficiency ranged from 31.3% to 47.6% in 24, 48, and 72 h after transformation. The green fluorescence mainly concentrated in the nucleus. With the increase of the expression of green fluorescence, granula was observed in the nucleus. RT-PCR and Western blotting analyses showed that pEGFP-alpha1-AGP had been integrated into the genome of Beijing chicken fibroblast cell with normal expression level. In optimized condition, there was no significant effect (P>0.05) on apoptosis ratio, positive cell shape, growth and reduplication state comparing with the control group. This research established the foundation for further function research of alpha1-AGP gene and application in transgenic animal cloning.
[show abstract][hide abstract] ABSTRACT: A fibroblast line (named SCF36) from ear marginal tissue of Simmental cattle was established successfully by direct culture of explants and cell cryopreservation techniques. Biological analysis showed that the population doubling time of the thawed cells was 42.8h. The average viability of the cells was 96.8% before freezing and 91.5% after thawing. Measurements of lactic dehydrogenase and malic dehydrogenase isoenzymes showed no cross-contamination of this cell line with other species. Karyotyping showed that the frequency of cells with chromosome number 2n=60 was more than 90%. Tests for bacteria, fungi, viruses and mycoplasmas were negative. The efficiencies of expression of enhanced green, yellow and red fluorescent protein genes (pEGFP-N(3), pEYFP-N(1) and pDsRed1-N(1)) were between 11.3% and 28.8% after transfection; fluorescence was well distributed in the cytoplasm and nucleus except for some cryptomeric vesicles. This Simmental cattle fibroblast line not only contains the germline of this important cattle breed, which is preserved at the cellular level, but valuable material has also been provided for genomic, postgenomic and somatic cloning research. Moreover, the establishment of these methods may provide both technical and theoretical support for preserving the genetic resources of other livestock and poultry at the cellular level.
[show abstract][hide abstract] ABSTRACT: Genetic polymorphisms of microsatellite locus BMS2508, which was closely linked to the ovine fecundity gene FecB, were detected in prolific (Small Tail Han sheep) and non-prolific breeds of sheep (Texel, Dorset and Chinese Merino). The linkage disequilibrium between microsatellite locus BMS2508 and FecB gene of Small Tail Han sheep was also analyzed. There was the same mutation (A746G) of BMPR-IB gene in Small Tail Han sheep as that of FecB in Booroola Merino ewes, but the FecB mutation was absent in Texel, Dorset and Chinese Merino sheep. The genotype frequencies of BB, B+ and ++ were 0.485, 0.398 and 0.117 in Small Tail Han sheep, respectively. There were eight alleles varied from 94 bp to 116 bp and 15 genotypes detected at BMS2508 locus in four sheep breeds totally 438 individual. The preponderant allele was 100 bp, 94 bp, 94 bp, 112 bp, 100 bp, 100 bp, 112 bp, and the frequency was 0.453, 0.544, 0.802, 0.475, 0.483, 0.439, 0.389 in Small Tail Han (n=307), Texel (n=45), Dorset (n=46), Chinese Merino (n=40), and BB group (n=149), B+ group (n=122), ++ group (n=36) from Small Tail Han, respectively. In Small Tail Han sheep, linkage analysis indicated that there was certain linkage disequilibrium between 100 bp allele of microsatellite BMS2508 and B allele of FecB gene (D' =0.408), and certain linkage disequilibrium between 110 bp and 114 bp alleles of microsatellite BMS2508 and + allele of FecB gene (D'=0.513).
[show abstract][hide abstract] ABSTRACT: A fibroblast line was successfully established from Mongolian horse ear marginal tissue by using a primary explant technique and cell cryogenic preservation technology. Biological analysis showed the following: The cells were adherent and exhibited density-dependent inhibition of proliferation; assays of microbial contamination from bacteria, fungi, and mycoplasma were negative; the population doubling time of the cells was 33.9 h; and a 2n chromosome number of 64 at a frequency higher than 80%. A lack of cross-contamination of this cell line with other species was confirmed by isoenzyme analysis of lactic and malic dehydrogenases. In order to study exogenous gene expression, four fluorescent proteins, pEGFP-N3, pEGFP-C1, pDsRed1-N1, and pEYFP-N1, were transfected into the cells. The corresponding fluorescence was distributed throughout the cytoplasm and nucleus 12 h after transfection. This cell line not only preserves the genetic resources of the Mongolian horse at the cellular level but also provides valuable materials for genomic, postgenomic, and somacloning research in this species.
[show abstract][hide abstract] ABSTRACT: The donor nucleus must experienced the epigenetic modification of the process reprogramming and went back to the initial state after the donor cell was injected into the oocytes. If the reprogramming is not completed, the efficiency of cloning will be reduced. However, reprogramming of nucleus muct was not only embodied in its ability after it was transferred into the oocytes. It was different in the potential if the cell type was not identical. In addition, different treatment to the donor cells resulted in different ability and the level of reprogramming. This paper described different effects of the type, algebra, cycles, age, and species of the donor cells after nuclear transplantation on the reprogramming. An overview of the exposition and analysis through the donor cell cryopreservation, serum starvation, and different reagent treatments were discussed.
[show abstract][hide abstract] ABSTRACT: PCR-RFLP was applied to analyze the polymorphism of MSTN gene UTR in 345 sheep that comprised of eleven sheep breeds, namely Texel sheep, Charolais sheep, Small-tailed Han sheep, Monggolian sheep, Ujumqin sheep, Altay Fat-rumped sheep, Hulunbeir sheep, Tashikurgan sheep, Duolang sheep, Hu sheep, and Gangba sheep. A 271 bp and a 1 003 bp long PCR products were digested with Mboand Bsato demonstrate polymorphism in the eleven sheep breeds, which were all at Hardy-Weinberg equilibrium (P>0.05). The distribution of 3 genotypes in 11 sheep breeds was significantly different (P<0.01). Digestion of the PCR products with HpyCH4 proved that 9 domestic local sheep breeds were different from Texel sheep in the SNP site that was associated with muscularity. The individual mutation base could generate the motifs for miRNA in the 3'UTR, and sequencing analysis demonstrated high frequency of mutation in the 3'UTR region.
[show abstract][hide abstract] ABSTRACT: The full-length of the cDNA sequence of Beijing fatty chicken ADSL gene was investigated by RT-PCR and RACE in this study. The results demonstrated that the complete ADSL cDNA revealed an open reading frame of 1455 nucleotides coding for 485 amino acid residues. The promoter of chicken ADSL cDNA showed typical features of house-keeping genes: no canonical TATA and CAAT boxes, 72.65% GC in 234 bp near the start codon, and there was a C-28T mutation, which caused the site CTCC mutating to the NRF-2 binding site CTTC. The complete open reading frame of the ADSL gene was inserted into expression vector pGEX-4T-1 and the fusion expression vector pGEX-ADSL was constructed. The pGEX-ADSL was transformed into Escherichia coli BL21 (DE3), positive cloning screened, induced and expresses by IPTG. The SDS-PAGE electrophoresis results showed that there were specific bands, about 80.5 kD and an isoelectric point of 6.79, which reached 26.9% of total cell proteins induced in 5hrs, and was an insoluble inclusion body. Under optimal conditions, ADSL was purified by Glutathione Sepharase 4B affinity chromatography, and Western blotting analysis showed that the fused protein was ADSL. This research was a foundation for further research into ADSL’s biological function and its application identification.
Zoological Research (ISSN: 0254-5853) Vol 29 Num 4. 01/2008;
[show abstract][hide abstract] ABSTRACT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetraz-olium bromide (MTT) assay was applied to measure the cell growth. And flow cytometry (FCM) was adopted to detect the changes of FRSs cell cycle and apoptosis rate .In addition, the Semiquantitative RT-PCR was using to assessed the regulation of dexamethasone(DEX) for Osteoprotegerin(OPG) or Ligand of receptor activator of nuclear factor kappa B(RANKL) in FRS. The results showed that TRAIL could prompt the growth of swine kidney fibroblast at the concentrations of 0.01–5 mg/L, but TRAIL inhibited the growth of FRS at the concentrations of 10–50 mg/L, but at the concentrations of 10–50 mg/L, TRAIL inhibited the growth of swine kidney fibroblast and even led them to apoptosis. DEX could regulate the effects of cytokines. The effect of DEX on TRAIL is synergies and the effective concentrations of DEX are 10-6–10-10 mol/L. Cell cycle analysis and apoptosis detection showed that the peak concentration of TRAIL inducing FRSs’ proliferation was 5 mg/L. Under this concentration, TRAIL(5 mg/L)+DEX (10-8 mol/L) group showed that the proliferation index of FRSs (Prl.) was increased by 2.49% compared with TRAIL 5 mg/L group(P<0.05); TRAIL10 mg/L showed that the proliferation index apoptosis rate of FRSs was not significant. But TRAIL(10 mg/L) + DEX(10-8 mol/L) group showed that the G0/G1 phase was increased by 2.36%(P<0.05) and the apoptosis rate was increased by 6.79%(P<0.01) compared with TRAIL 10 mg/L group. Semi-quantitative RT-PCR showed that DEX could inhibit the constitutive OPG and stimulate RANKL mRNA steady-state levels in a dose-dependent manner. These results indicate that TRAIL prompt not only the growth but also the apoptosis of FRSs which depends on the concentrations of TRAIL. The phenomenon is related to the change of cell cycle of FRSs induced by TRAIL and DEX. The OPG/RANK/RANKL system is an important pathway that DEX can mediate the effects of TRAIL on proliferation and apoptosis of FRSs[Acta Zoologica Sinica 54(5): 836 – 845, 2008].
[show abstract][hide abstract] ABSTRACT: With the establishment of modern sheep production systems in China, various forms of hybridization with Western breeds and
between native breeds have been utilized for genetic improvement. At the same time, the progressive destruction or deterioration
of sheep habitat has accompanied urbanization in China. Together these factors have accelerated the loss of genetic diversity,
or even resulted in the extinction of some indigenous breeds. It is therefore important that efficient strategies for surveillance,
evaluation, conservation and utilization of available genetic resources are developed for this species. In this study, a total
of 30 microsatellite markers were used to assess genetic diversity for 12 native breeds and one Western sheep breed in Northern
China. The high polymorphism information contents at the 30 markers, varying from averages of 0.519 to 0.666 for the 13 breeds,
imply the retention of natural variation from source populations in the domestic breeds from different geographic regions
in China. Analysis of genetic differentiation revealed substantial divergence among these breeds. Neutrality tests indicated
that more than one third of the 30 loci were in departure from neutrality, implying that some evolutionary forces (e.g. selection
and migration) had acted on these populations. Phylogenetic and phylogeographic analyses displayed a remarkable degree of
consistency between geographic origins, breeding histories and the pattern of genetic differentiation.
[show abstract][hide abstract] ABSTRACT: The genetic variations of microsatellites in intron 2 of the porcine adipocyte fatty acid-binding protein (A-FABP) genes were investigated in 420 pigs including Wuzhishan pig, Yimeng black pig, Hanjiang black pig, Laiwu pig, Beijing black pig, Min pig, Chenghua pig, Neijiang pig, Erhualian pig, Bama xiang pig and Large White pig. The results suggested as follows: (i)PIC of the Wuzhishan pig breed is the highest (0.7904) and 11 alleles were detected. Compared with Large White pig,Chinese pig breeds showed a great polymorphism of A-FABP microsatellites except Beijing black pig in which only 2 alleles were detected. (ii) Only Min pig, Bama xiang pig, Beijing black pig and Large White pig were in Hardy-Weinberg equilibrium. (iii) The analysis of genetic differentiation showed that the average value of the A-FABP gene differentiation of 10 Chinese pigs is about 40.83%.