• 한국축산식품학회지
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• 제38권4호
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• pp.816-822
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• 2018

Bifidobacterium is recognized as one of the most beneficial microorganisms in our gut. Many researches on bifidobacteria have been done to understand their roles in the gut. The objective of the present study was to develop a bioluminescent labelling plasmid vector for bifidobacteria to facilitate their visualization in vitro, in situ, and in vivo. A plasmid replicon (2.0 kb) of plasmid pFI2576 previously identified from B. longum FI10564 was amplified by PCR and cloned into pUC19 plasmid vector (2.68 kb). The cloned replicon was subcloned into pTG262 ($luc^+$) recombinant plasmid vector (7.4 kb) where a luciferase gene ($luc^+$) from pLuc2 (8.5 kb), an Escherichia coli and lactobacilli shuttle vector, was inserted into pTG262 plasmid vector. The final recombinant DNA, pTG262::pFI2576 rep ($luc^+$), was transferred into a B. catenulatum strain. This recombinant strain showed 3,024 relative luminescence units at $OD_{600}$ value of 0.352. Thus, this recombinant plasmid construct can be broadly used for labelling bifidobacteria.

• Asian-Australasian Journal of Animal Sciences
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• 제26권11호
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• pp.1644-1650
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• 2013

Ribosomal protein (rp) S6 is the substrate of ribosomal protein S6K (S6 kinase) and is involved in protein synthesis by mTOR/S6K/S6 signaling pathway. Some S6 cDNA have been cloned in mammals in recent years but has not been identified in the goat. To facilitate such studies, we cloned the cDNA encoding Cashmere goat (Capra hircus) S6 (GenBank accession GU131122) and then detected mRNA expression in seven tissues by real time PCR and protein expression in testis tissue by immunohistochemisty. Sequence analysis indicated that the obtained goat S6 was a 808 bp product, including a 3' untranslated region of 58 bp and an open reading frame of 750 bp which predicted a protein of 249 amino acids. The predicted amino acid sequence was highly homologous to cattle, human, mouse and rat S6. Expression analysis indicated S6 mRNA was expressed extensively in detected tissues and S6 protein was expressed in testis tissue.

• Asian-Australasian Journal of Animal Sciences
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• 제18권12호
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• pp.1684-1690
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• 2005

Using mRNA differential display technique, we investigated differential gene expression in hybrids relative to their parents in a diallel cross involving four chicken breeds in order to provide an insight into the molecular basis of heterosis in chicken. The results indicated that there was extensive differential gene expression between chicken F1 hybrids and their parents which was classified into four kinds of patterns as following: (1) bands only detected in hybrid F1; (2) bands only absent in hybrid F1; (3) bands only detected in parent P1 or P2; (4) bands absent in parent P1 or P2. Forty-two differentially expressed cDNAs were cloned and sequenced, and their expression patterns were confirmed by Reverse-Northern dot blot. Sequence analysis and database searches revealed that genes showed differential expression between hybrid and parents were regulatory and functional genes involved in metabolism, mRNA splicing, transcriptional regulation, cell cycles and protein modification. These results indicated that hybridization between two parents can cause changes in expression of a variety of genes. In conclusion, that the altered pattern of gene expression in hybrids may be responsible for heterosis in chickens.

• Reproductive and Developmental Biology
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• 제35권1호
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• pp.77-83
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• 2011

One-step dilution and direct transfer would be a practical technique for the field application of frozen embryo. This study was to examine whether Jeju Black Cattle (JBC, Korean Cattle) can be successfully cloned from vitrified and one-tep diluted somatic cell nuclear transfer (SCNT) blastocyst after direct transfer. For vitrification, JBC-SCNT blastocysts were serially exposed in glycerol (G) and ethylene glycol (EG) mixtures [10%, (v/v) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.] which is diluted in 10% FBS added D-PBS. And then SCNT blastocysts were loaded in 0.25 ml mini straw, placed in cold nitrogen vapor for 3 min. and then plunged into $LN_2$. One-step dilution in straw was done in $25^{\circ}C$ water for 1 min, by placing vertically in the state of plugged-end up and down for 0.5 min, respectively. When in vitro developmental capacity of vitrified SCNT blastocyst was examined at 48 h after one-step dilution, hatched rate (56.4%) was slightly lower than that of control group (62.5%). In field trial, when the vitrified-thawed SCNT blastocysts were transferred into uterus of synchronized 5 recipients, a cloned female JBC was delivered by natural birth on day 299 and healthy at present. In addition, when the short tandem repeat marker analysis of the cloned JBC was evaluated, microsatellite loci of 11 numbers was perfectly matched genotype with donor cell (BK94-14). This study suggested that our developed vitrification and one-step dilution technique can be applied effectively on field trial for cloned animal production, which is even no longer in existence.

• Animal Bioscience
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• 제34권5호
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• pp.801-810
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• 2021

Objective: microRNAs (miRNAs) can play a role in a variety of physiological and pathological processes, and their role is achieved by regulating the expression of target genes. Our previous high-throughput sequencing found that ssc-miR-185 plays an important regulatory role in piglet diarrhea, but its specific target genes and functions in intestinal porcine epithelial cell (IPEC-J2) are still unclear. We intended to verify the target relationship between porcine miR-185 and cell division cycle 42 (CDC42) gene in IPEC-J2 and to explore the effect of miR-185 on the proliferation of IPEC-J2 cells. Methods: The TargetScan, miRDB, and miRanda software were used to predict the target genes of porcine miR-185, and CDC42 was selected as a candidate target gene. The CDC42-3' UTR-wild type (WT) and CDC42-3'UTR-mutant type (MUT) segments were successfully cloned into pmirGLO luciferase vector, and the luciferase activity was detected after co-transfection with miR-185 mimics and pmirGLO-CDC42-3'UTR. The expression level of CDC42 was analyzed using quantitative polymerase chain reaction and Western blot. The proliferation of IPEC-J2 was detected using cell counting kit-8 (CCK-8), methylthiazolyldiphenyl-tetrazolium bromide (MTT), and 5-ethynyl-2'-deoxyuridine (EdU) assays. Results: Double enzyme digestion and sequencing confirmed that CDC42-3'UTR-WT and CDC42-3'UTR-MUT were successfully cloned into pmirGLO luciferase reporter vector, and the luciferase activity was significantly reduced after co-transfection with miR-185 mimics and CDC42-3'UTR-WT. Further we found that the mRNA and protein expression level of CDC42 were down-regulated after transfection with miR-185 mimics, while the opposite trend was observed after transfection with miR-185 inhibitor (p<0.01). In addition, the CCK-8, MTT, and EdU results demonstrated that miR-185 promotes IPEC-J2 cells proliferation by targeting CDC42. Conclusion: These findings indicate that porcine miR-185 can directly target CDC42 and promote the proliferation of IPEC-J2 cells. However, the detailed regulatory mechanism of miR-185/CDC42 axis in piglets' resistance to diarrhea is yet to be elucidated in further investigation.

• 대한수의학회지
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• 제52권1호
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• pp.25-31
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• 2012

Brucella (B.) canis is mainly transmitted by direct or indirect contact with aborted fetuses and placenta. It's also known to be able to infect human, which likely results in providing veterinarians and companion animal owners for infectious risk. To develop diagnostic ELISA, we cloned and expressed rp1L gene of B. canis, which encodes the ribosomal protein L7/L12. Using this purified recombinant protein, indirect-ELISA (iELISA) was evaluated using 78 positive and 44 negative sera. The sensitivity and the specificity of iELISA were 94% and 89%, respectively. The results indicated that indirect-ELISA using recombinant ribosomal protein L7/L12 may be useful for diagnosis of canine brucellosis.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 발생공학 국제심포지움 및 학술대회 발표자료집
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• pp.25-31
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• 2001

During early development, a dramatic reduction in methylation levels occurs in mouse (Monk et al., 1987). The process of epigenetic reprogramming in early embryos erases gamete-specific methylation patterns inherited from the parents (Howlett ＆ Reik 1991, Monk et al., 1987, Oswald et al., 2000, Sanford et al., 1984). This genome-wide demethylation process may be a prerequisite for the formation of pluripotent stem cells that are important for the later development (Reik ＆ Surani 1997). During post-implantation development, a wave of de novo methylation takes place; most of the genomic DNA is methylated at defined developmental timepoints, whereas tissue-specific genes undergo demethylation in their tissues of expression (Kafri et al., 1992, Razin ＆ Kafri 1994). Another demethylation-remethylation cycle of epigenetic reprogramming takes place during gametogenesis and is necessary for resetting of genomic imprinting (Solter 1988). The dynamic epigenetic reprogramming events appear to be basic and are probably conserved in eutherian mammals (see below). (omitted)

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.83-90
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• 2007

Insulin-like growth factor II (IGF2) and H19 genes are mutually imprinted genes which may be responsible for abnormalities in the cloned fetuses and offspring. This study was performed to identify putative differentially methylated regions (DMRs) of porcine H19 locus and to explore its genomic imprinting in in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) embryos. Based on mice genomic data, we identified DMRs on H19 and found porcine H19 DMRs that included three CTCF binding sites. Methylation patterns in IVF and SCNT embryos at the 2-, 4-, $8{\sim}16$-cells and blastocyst stages were analyzed by BS (Bisulfite Sequencing)-PCR. The CpGs in CTCF1 was significantly unmethylated in the 2-cell stage IVF embryos. However, the 4- (29.1%) and $8{\sim}16$-cell (68.2%) and blastocyst (48.2%) stages showed higher methylation levels (p<0.01). On the other hand, SCNT embryos were unmethylayted ($0{\sim}2%$) at all stages of development. The CpGs in CTCF2 showed almost unmethylation levels at the 2-,4- and $8{\sim}16$-cell and blastocyst stages of development in both IVF ($0{\sim}14.1%$) and SCNT ($0{\sim}6.4%$) embryos. At all stages of development, CTCF3 was unmethylated in IVF ($0{\sim}17.3%$) and SCNT ($0{\sim}1.2%$) embryos except at the blastocyst stage (54.5%) of IVF embryos. In conclusion, porcine SCNT embryos showed an aberrant methylation pattern comprised to IVF embryos. Therefore, we suggest that the aberrant methylation pattern of H19 loci may be a reason for increased abnormal fetus after embryo transfer of porcine SCNT embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제27권6호
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• pp.778-783
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• 2014

The polymorphism of microphthalmia-associated transcription factor (MITF) and tyrosinase (TYR) genes have been proposed to play a vital role in coat colour genesis in mammals, but their role remains ambiguous in geese at best. Here, we cloned and sequenced 1,397 bp coding region of MITF gene and a 588 bp fragment of TYR exon 1 for polymorphism analysis among 157 domestic geese showing three types of plumage colour. We detected a total of three SNPs (c.280T>C, c.345G>A, and c.369G>A) in TYR and six haplotypes (H1-H6). Among them, haplotypes H1, H2, H3, and H5 were significantly associated with white plumage trait of Zhedong White Geese. However, only diplotype H1H1 and H3H5 were significantly associated with white plumage trait of Zhedong White Geese (p<0.01). We only detected one SNP (c.1109C>T) for MITF gene and found that genotype CT and TT were significantly associated with white plumage trait of Zhedong White Geese. Briefly, our study suggested an association between polymorphisms of TYR and MITF genes and the plumage colour trait in domestic geese.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.186-186
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• 2004

In this study, we generated 40 somatic cell cloned (scNT) piglets. Of these, three displayed congestion in both liver and lung, and died within the first week of life. Two-dimensional gel electrophoresis experiments revealed changes in the responses of several detoxification-related proteins to stress and inflammation. As a result, congestive livers and lungs displayed extensive hepatopneumonic apoptosis.(omitted)