• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.55-55
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• 2003

The random insertion of useful gene in genome has been a common method to produce transgenic animals. This method is inefficient for induction of high levels gene expression in transgenic animals. To improve this limit, we tried to develop the system which target the gene at the specific genomic region. Thus, in our experiment, the vector system to target the human thrombopoietin (TPO) gene was developed. Targeting vector including TPO, neo and DT genes was transfrcted into bovine embryonic fibroblasts (bEF) or bovine ear skin fibroblasts (bESF). First of all, we determined concentration of the geneticin (G418) for selection of transfected cell lines. Our results showed that 1200 and 900 $\mu\textrm{g}$/ml of G418 were the most proper for selection of transfscted bEF and bESF cells. In this study, lipofectamine was used as a transfection reagent. Thus, the proper ratio of DNA:lipofectamine for transfection was also required to elevate targeting efficiency in primary mammalian cells. Our result indicates that the most proper ratios of DNA:lipofectamine were 4:2 and 1:2 in bEF and bESF cells. According to the optimized these conditions, single colonies were picked following transfection and were analyzed by PCR. More than 90% of the single colonies have TPO gene. However, there were no colonies with targeted TPO at the specific genomic region. Therefore, further experiments to select the specifically targeted colonies and to find more efficient methods such as reducing selection time and shortening a size of TPO gene are required.

• Korean Journal of Veterinary Service
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• v.30 no.3
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• pp.393-406
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• 2007

Bovine tuberculosis is an important zoonosis worldwide. Mycobacterium bovis, the causative agent of this disease in cattle, is also a pathogen for humans and several economically important animals. The cases of tuberculosis are reported in two cow found at slaughter house located in Gwangju city. Histopathologically, in the lymph nodes, granulomas consisted of large areas of necrosis surrounded by variable thick bands of cellular infiltrate containing macrophages, Langhans-type multinucleated giant cells and lymphocytes. Lesions in the lung followed the same developmental pattern as did lesions in the lymph nodes with some exceptions. With the acid-fast staining, numerous mycobacteria were revealed in the lung and lymph nodes. M bovis was confirmed as a causative agent in these cattle using bacterial isolation and PCR and restriction fragment length polymorphism method based on a unique 12.7 kb fragment insertion sequence from the Mycobacterium tuberculosis genome and the pncA polymorphism, The insertion element IS6110 and IS1081 were present M bovis isolated from lungs and lymph nodes of cattle using PCR assay. These cases are interesting and important in public health aspect that M bovis-infected cattle were found during a routine post-mortem inspection at slaughter house.

• Korean Journal of Microbiology
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• v.38 no.2
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• pp.86-95
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• 2002

As a result of genome projects, the research to elucidate the function of a protein of interest has recently been well-recognized. In order to facilitate functional genomics, a useful mammalian gene expression vector is required. Using an infectious CDNA clone of BVDV pNADLclns-, we have developed a mammalian gene expression vector. In this study, a replication-competent full-length infectious CDNA clone containing puremycin acetyltransferase (pac) gene (pNADLclns-/pac) was successfully generated. The viral RNA replication and viral protein NS3 synthesis were examined by detecting metabollically $^{32}P$-labelled genomic viral RNA and immunoblotting with a mouse anti-NS3 antibody. To generate viral replicon as an expression vector, we examine if the viral structural genes (C, E0, El, E2) are required for viral replication by deletion analysis. As a result, all of the structural proteins are dispensable for viral replication per se, but essential for infectious viral particle formation. Based on our deletion analysis, we have generated a replication-competent BVDV viral replicon (pNADLclns-/pac/${\Delta}S$), whose structural genes are all deleted. In addition to NADLclns- /pac/${\Delta}S$, NADLclns-/ luc/${\Delta}S$ viral replicon containing luciferase gene as a reporter was constructed and fecund to be replication-compotent in HeLa and BHK cells as well as MDBK cells. Therefore, BVDV viral replicon developed in our study will be a useful tool to express a protein of interest in various mammalian cells.

• Journal of Animal Science and Technology
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• v.53 no.3
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• pp.195-202
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• 2011

Maintenance of adequate telomere length in developing cells is the most important concern to preserve the integrity of the genome. The length of telomere is strictly regulated by numerous telomere-binding proteins and/or interacting factors. Even though the expression of telomerase in the male reproductive tract has been characterized, developmental expressional profiling of telomerase and other telomere-associated proteins has not been determined in detail. The present study was attempted to examine expression patterns of catalytic subunit (Tert) and RNA component (Terc) of telomerase and two telomerase associated factors, telomerase associated protein 1 (Tep1) and TERF1 (TRF1) interacting nuclear factor 2 (Tinf2) in the testis and seminal vesicle of male rat during postnatal development. The real-time PCR analysis was utilized to quantify mRNA expression of molecules. The abundance of Tep1 mRNA in the testis and seminal vesicle was the highest at 5 months of age. Expressional fluctuation of Tinf2 during postnatal development was found in the testis, while expression of Tinf2 in the seminal vesicle was gradually increased until 5 months of age and then significantly decreased later. mRNA level of Tert gene in the testis was significantly increased at the adult and the elder, while the highest expression of Tert gene in the seminal vesicle was found at 5 months of age. Expression of Terc transcript in the testis and seminal vesicle was the highest at 5 months of age, followed by significant reduction at 1 and 2 years of ages. Such differential gene expression of telomere-associated factors and telomerase components in different male reproductive tissues during postnatal development indicates that maintenance of telomere length would be regulated in tissue- and/or age-specific manners.

• Journal of Animal Science and Technology
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• v.57 no.12
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• pp.47.1-47.10
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• 2015

Background: Knowledge of linkage disequilibrium (LD) levels among different populations can be used to detect genetic diversity and to investigate the historical changes in population sizes. Availability of large numbers of SNP through new sequencing technologies has provided opportunities for extensive researches in quantifying LD patterns in cattle breeds. The aim of this study was to compare the extent of linkage disequilibrium among Iranian cattle breeds using high density SNP genotyping data. Results: A total of 70 samples, representing seven Iranian indigenous cattle breeds, were genotyped for 777962 SNPs. The average values of LD based on the $r^2$ criterion were computed by grouping all syntenic SNP pairwises for intermarker distances from 0 Kb up to 1 Mb using three distance sets. Average $r^2$ above 0.3 was observed at distances less than 30 Kb for Sistani and Kermani, 20 Kb for Najdi, Taleshi, Kurdi and Sarabi, and 10 Kb for Mazandarani. The LD levels were considerably different among the Iranian cattle breeds and the difference in LD extent was more detectable between the studied breeds at longer distances. Lower level of LD was observed for Mazandarani breed as compared to other breeds indicating larger ancestral population size in this breed. Kermani breed continued to have more slowly LD decay than all of the other breeds after 3 Kb distances. More slowly LD decay was observed in Kurdi and Sarabi breeds at larger distances (>100 Kb) showing that population decline has been more intense in more recent generations for these populations. Conclusions: A wide genetic diversity and different historical background were well reflected in the LD levels among Iranian cattle breeds. More LD fluctuation was observed in the shorter distances (less than 10 Kb) in different cattle populations. Despite of the sample size effects, High LD levels found in this study were in accordance with the presence of inbreeding and population decline in Iranian cattle breeds.

• Korean Journal of Animal Reproduction
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• v.27 no.4
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• pp.309-315
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• 2003

DNA methylation at CpG sites, which is a epigenetic modification, is associated with gene expression without change of DNA sequences. During early mouse embryogenesis, dynamic changes of DNA methylation occur. In this study, DNA methylation patterns of porcine embryos produced in vivo and in vitro were examined at various developmental stages by the immunocytochemical staining method. Interestingly, active demethylation was not observed on the paternal pronucleus of porcine zygotes. However, differences were detected in the passive demethylation process between in vivo and in vitro embryos. There was no change in the DNA methylation state until the blastocyst stage of in vivo embryos, whereas partial demethylation was observed in several blastomeres from a 4 cell stage to a morula stage of in vitro embryos. The whole genome of inner cell mass (ICM) and trophectoderm (TE) cells in porcine blastocysts were evenly methylated without de novo methylation. Our findings demonstrate that genome-wide demethylation does not occur in pig embryos during preimplantation development unlike murine and bovine embryos. It indicates that the machinery regulating epigenetic reprogramming may be different between species.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.10
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• pp.1381-1386
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• 2014

In spite of variation in coat color, size, and production traits among indigenous Bangladeshi cattle populations, genetic differences among most of the populations have not been investigated or exploited. In this study, we used a high-density bovine single nucleotide polymorphism (SNP) 80K Bead Chip derived from Bos indicus breeds to assess genetic diversity and population structure of 2 Bangladeshi zebu cattle populations (red Chittagong, n = 28 and non-descript deshi, n = 28) and a semi-domesticated population (gayal, n = 17). Overall, 95% and 58% of the total SNPs (69,804) showed polymorphisms in the zebu and gayal populations, respectively. Similarly, the average minor allele frequency value was as high 0.29 in zebu and as low as 0.09 in gayal. The mean expected heterozygosity varied from $0.42{\pm}0.14$ in zebu to $0.148{\pm}0.14$ in gayal with significant heterozygosity deficiency of 0.06 ($F_{IS}$) in the latter. Coancestry estimations revealed that the two zebu populations are weakly differentiated, with over 99% of the total genetic variation retained within populations and less than 1% accounted for between populations. Conversely, strong genetic differentiation ($F_{ST}=0.33$) was observed between zebu and gayal populations. Results of population structure and principal component analyses suggest that gayal is distinct from Bos indicus and that the two zebu populations were weakly structured. This study provides basic information about the genetic diversity and structure of Bangladeshi cattle and the semi-domesticated gayal population that can be used for future appraisal of breed utilization and management strategies.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.5
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• pp.603-608
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• 2013

Hanwoo have been subjected over the last seventy years to intensive artificial selection with the aim of improving meat production traits such as marbling and carcass weight. In this study, we performed a signature of selection analysis to identify recent positive selected regions driven by a long-term artificial selection process called a breeding program using whole genome SNP data. In order to investigate homozygous regions across the genome, we estimated iES (integrated Extended Haplotype Homozygosity SNP) for the each SNPs. As a result, we identified two highly homozygous regions that seem to be strong and/or recent positive selection. Five genes (DPH5, OLFM3, S1PR1, LRRN1 and CRBN) were included in this region. To go further in the interpretation of the observed signatures of selection, we subsequently concentrated on the annotation of differentiated genes defined according to the iES value of SNPs localized close or within them. We also described the detection of the adaptive evolution at the molecular level for the genes of interest. As a result, this analysis also led to the identification of OLFM3 as having a strong signal of selection in bovine lineage. The results of this study indicate that artificial selection which might have targeted most of these genes was mainly oriented towards improvement of meat production.

• Journal of Animal Science and Technology
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• v.62 no.2
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• pp.141-158
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• 2020

Skin is a major thermoregulatory organ in the body controlling homeothermy, a critical function for climate adaptation. We compared genes expressed between tropical- and temperate-adapted cattle to better understand genes involved in climate adaptation and hence thermoregulation. We profiled the skin of representative tropical and temperate cattle using RNA-seq. A total of 214,754,759 reads were generated and assembled into 72,993,478 reads and were mapped to unique regions in the bovine genome. Gene coverage of unique regions of the reference genome showed that of 24,616 genes, only 13,130 genes (53.34%) displayed more than one count per million reads for at least two libraries and were considered suitable for downstream analyses. Our results revealed that of 255 genes expressed differentially, 98 genes were upregulated in tropically-adapted White Fulani (WF; Bos indicus) and 157 genes were down regulated in WF compared to Angus, AG (Bos taurus). Fifteen pathways were identified from the differential gene sets through gene ontology and pathway analyses. These include the significantly enriched melanin metabolic process, proteinaceous extracellular matrix, inflammatory response, defense response, calcium ion binding and response to wounding. Quantitative PCR was used to validate six representative genes which are associated with skin thermoregulation and epithelia dysfunction (mean correlation 0.92; p < 0.001). Our results contribute to identifying genes and understanding molecular mechanisms of skin thermoregulation that may influence strategic genomic selection in cattle to withstand climate adaptation, microbial invasion and mechanical damage.

• Genomics & Informatics
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• v.13 no.4
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• pp.146-151
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• 2015

Previous studies in Holstein have shown 35% to 51.8% heritability in milk production traits, such as milk yield, fat, and protein, using pedigree data. Other studies in complex human traits could be captured by common single-nucleotide polymorphisms (SNPs), and their genetic variations, attributed to chromosomes, are in proportion to their length. Using genome-wide estimation and partitioning approaches, we analyzed three quantitative Holstein traits relevant to milk production in Korean Holstein data harvested from 462 individuals genotyped for 54,609 SNPs. For all three traits (milk yield, fat, and protein), we estimated a nominally significant (p = 0.1) proportion of variance explained by all SNPs on the Illumina BovineSNP50 Beadchip ($h^2_G$). These common SNPs explained approximately most of the narrow-sense heritability. Longer genomic regions tended to provide more phenotypic variation information, with a correlation of 0.46~0.53 between the estimate of variance explained by individual chromosomes and their physical length. These results suggested that polygenicity was ubiquitous for Holstein milk production traits. These results will expand our knowledge on recent animal breeding, such as genomic selection in Holstein.