• Development and Reproduction
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• v.27 no.4
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• pp.205-211
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• 2023

INTS14/VWA9, a component of the integrator complex subunits, plays a pivotal role in regulating the fate of numerous nascent RNAs transcribed by RNA polymerase II, particularly in the biogenesis of small nuclear RNAs and enhancer RNAs. Despite its significance, a comprehensive mutation model for developmental research has been lacking. To address this gap, we aimed to investigate the expression patterns of INTS14 during zebrafish embryonic development. We generated ints14 mutant strains using the CRISPR/Cas9 system. We validated the gRNA activity by co-injecting Cas9 protein and a single guide RNA into fertilized zebrafish eggs, subsequently confirming the presence of a 6- or 9-bp deletion in the ints14 gene. In addition, we examined the two mutant alleles through PCR analysis, T7E1 assay, TA-cloning, and sequencing. For the first time, we used the CRISPR/Cas9 system to create a model in which some sequences of the ints14 gene were removed. This breakthrough opens new avenues for in-depth exploration of the role of ints14 in animal diseases. The mutant strains generated in this study can provide a valuable resource for further investigations into the specific consequences of ints14 gene deletion during zebrafish development. This research establishes a foundation for future studies exploring the molecular mechanisms underlying the functions of ints14, its interactions with other genes or proteins, and its broader implications for biological processes.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.1
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• pp.7-12
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• 2010

The full-length cDNA of the porcine peroxisomal ${\Delta}^3$,${\Delta}^2$-enoyl-CoA isomerase (PECI) gene encodes a monofunctional peroxisomal ${\Delta}^3$,${\Delta}^2$-enoyl-CoA isomerase. Cloning and sequencing of the porcine PECI cDNA revealed the presence of an 1185-base pair open reading frame predicted to encode a 394-amino acid protein by the 5'rapid amplification of cDNA ends (5'RACE) and EST sequences. The porcine PECI gene was expressed in seven tissues (heart, liver, spleen, lung, kidney, skeletal muscle, fat) which was revealed by reverse transcriptase-polymerase chain reaction (RT-PCR). The porcine PECI was mapped to SSC71/2 p11-13 using the somatic cell hybrid panel (SCHP) and the radiation hybrid panel (RH) (LOD score 12.84). The data showed that PECI was closely linked to marker S0383. A C/T single nucleotide polymorphism in PECI exon 10 (3'UTR) was detected as a PvuII PCR-RFLP. Association analysis in our experimental pig population showed that different genotypes of PECI gene were significantly associated with the Average Backfat thickness (ABF) (p<0.05) and Buttock backfat thickness (p<0.01).

• Journal of Animal Science and Technology
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• v.50 no.1
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• pp.9-18
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• 2008

Species-specific polymorphisms in chicken, pheasant, turkey, and quail were identified by cloning and sequencing of the immunoglobulin constant domain (IgLC). A set of species-specific primers were then designed on the basis of polymorphisms in the IgLC between species, as well as two additional sets of primers for the cytochrome b and tapasin genes, for the purpose of species identification. Together, the primers successfully distinguished specific species from chicken by species-specific PCR. This simple but unambiguous method may be used to screen avian inter-species germline chimeras, which are valuable models for the conservation of endangered species.

• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.879-887
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• 2014

Bacteria that mediate the anaerobic oxidation of ammonium (anammox) have been detected in natural ecosystems, as well as various wastewater treatment systems. In this study, sludge from a particular landfill leachate anaerobic treatment system was selected as the incubation seed for anammox microorganism enrichment owing to its possible anammox activity. Transmission electron microscopy observation, denaturing gradient gel electrophoresis analysis, and cloning/sequencing techniques were applied to identify the diversity of anammox microorganisms throughout the incubation. During the early stage of operation, the diversity of anammox microorganisms was similar to the original complex microbes in the seed sludge. However, as incubation time increased, the anammox microorganism diversity within the system that was originally dominated by Candidatus (Ca.) Brocadia sp. was replaced by Ca. Anammoxoglobus propionicus. The domination of Ca. Anammoxoglobus propionicus produced a stable removal of ammonia (70 mg-N/l) and nitrite (90 mg-N/l), and the total nitrogen removal efficiency was maintained at nearly 95%. The fluorescence in situ hybridization results showed that Ca. Anammoxoglobus propionicus was successfully enriched from $1.8{\pm}0.6%$ initially to $65{\pm}5%$ after 481 days of operation. Therefore, the present results demonstrated the feasibility of enriching Ca. Anammoxoglobus propionicus from leachate sludge, even though the original cell count was extremely low. Application of this seldom found anammox organism could offer an alternative to current ammonia-nitrogen treatment.

• Journal of fish pathology
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• v.7 no.2
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• pp.95-103
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• 1994

We have prepared cDNA libray of loach. M. mizolepis in order to isolate cDNA clone of growth hormone gene. Total RNA was isolated from pituitary of loach, and then mRNA was further purified from total RNA by oligo (dT)-coupled magnetic beads. The purified mRNA was used as substrates to prepare cDNA. The resulting cDNA was ligated into the EcoRV/Smal site of pBlueKS+. The ligation mixture have transformed E. coli JM109 strain with electroporator to obtain high yield of transformation efficiency. All the transformants was screened with DIG-labeled Tilapia growth hormone gene by high density colony hybridization. After isolating 10 putative colonies showing the positive signals, secondary colony hybridization and southern hybridization could confirm it as true clones. The nucleotide sequence of one candidate, pCGHI, was compared with 312 bp DNA fragment used as DNA probe and show 52% relative homology to Tilapia growth hormone gene.

• Journal of Animal Science and Technology
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• v.46 no.3
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• pp.301-306
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• 2004

This study was conducted to develop new markers at the region that was related to QTL affecting intramuscular fat and backfat thickness on chromosome 6q28 - 6q32 in pigs. Dozens of repeated sequences were founded using shotgun sequencing of several BAC clones corresponding to that region, of which five new microstellite markers that identified polymorphism were discovered. The mean number of alleles at each locus observed 2.13(KP0290F2), 4.63(KP0248Cll), 7.38(KP1231C91), 2.75(KPI23IC92) and 6.2S(KP1231C93) in 8 breeds(Landrace, Korean native pig, Duroc, Yorkshire, Berkshire, Wuzhishan pig, Xiang pig, Min pig). The average estimated heterozygosity values at each locus varied from 0.2100(KP0290F2) to 0.8304(KPI23IC91) in all populations. In other hand, the average allele of all loci WlL'I within range of 0.4517(Berkshire) and 0.6957 (Yorkshire). Of these markers, KP0248C11, KP1231C91 and KP1231C93 were identified to have optimal number of alleles, high heterozygosity values and low standard deviation values. Especially, KPI23IC91 and KPI231C93 might be considered as a useful marker for genetic mapping and diversity study.

• Microbiology and Biotechnology Letters
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• v.33 no.2
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• pp.96-105
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• 2005

For screening of autoregulator receptor gene from Streptomyces longwoodensis, PCR was performed with primers of receptor gene designed on the basis of amino acid sequences of autoregulator receptor proteins with known function. PCR products were subcloned into the BamHIsite of pUC19 and transformed into the E. coli $DH5{\alpha}$. The isolated plasmid from transformant contained the fragment of 100 bp, which was detected on $2\%$ gel after BamHI treatment. The insert, 100 bp PCR product, was confirmed as the expected internal segment of gene encoding autoregulator receptor protein by sequencing. Southern and colony hybridizations with the 100 bp fragment as a probe allowed to select a genomic clone of S. longwoodensis, pSLT harboring a 4.4 kb SphI fragment. Nucleotide sequencing analyses revealed a 651 bp open reading frame(ORF) were isolated protein showing moderate homology ($35{\sim}46\%$) with the ${\Gamma}$-butyrolactone autoregulator receptors from Streptomyces sp., and this ORF was named sltR The sltR/pET-17b plasmid was constructed to overexpress the recombinant SltR protein (rSltR) in E. coli BL21 (DE3)/pLysS, and the rSltR protein was purified to homogeneity by DEAE-Sephacel column chromatography, and DEAE-5PW chromatography (HPLC). The molecular mass of the purified rSltR protein was 55 kDa by HPLC gel-filtration chromatography and 28 kDa by SDS-PAGE, indicating that the rSltR protein is present as a dimer. A binding assay with tritium-labeled autoregulators revealed that the rSltR has clear binding activity with a A-factor type autoregulator as the most effective ligand.

• Journal of Veterinary Science
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• v.21 no.5
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• pp.64.1-64.14
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• 2020

Background: Canine distemper virus (CDV) infection results in high morbidity and mortality in dogs. There has been no report about Isolation of Korean CDV since 1980 in Korea. Objectives: To investigate the biological properties and the genetic characterization of Korean CDV. Methods: Vero cells expressing dog signaling lymphocyte activation molecule (dSLAM) gene named as Vero/dSLAM were used to isolate CDV using 17 samples. Diagnostic methods such as cytopathic effects, immunofluorescence assay, peroxidase linked assay, electron microscopy, rapid immunodiagnostic assay, and reverse transcription polymerase chain reaction were used to confirm the Korean CDV isolate as a CDV. The genetic analysis was performed through cloning and sequencing of hemagglutinin gene of CDV isolate. Results: A virus propagated in Vero/dSLAM cell was confirmed as CDV (CD1901 strain) based on the above methods. The CD1901 strain showed the highest viral titer (10^5.5 50% tissue culture infectious dose [TCID₅₀]/mL) in the Vero/dSLAM cells at 4 days post inoculation, but did not form a fork on chorioallantoic membrane of 7-day-old egg. Ribavirin, a nucleotide analogue anti-viral agent, inhibits moderately the Korean CDV propagation in the Vero/dSLAM cells. The nucleotide and amino acid sequences of the H gene of CD1901 strain were compared with those of other CDV strains. The CD1901 strain belonged to Asia 1 group and had the highest similarity (99.9%) with the BA134 strain, which was isolated in China in 2008. Conclusions: We constructed successfully Vero/dSLAM and isolated one Korean CDV isolate (CD1901 strain) from a naturally infected dog. The CD1901 strain belonged to Asia 1 genotype.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.10
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• pp.1581-1590
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• 2018

Objective: The aim of this study was to clone alternative splicing isoforms of pig myoneurin (MYNN), predict the structure and function of coding protein, and study temporal and spatial expression characteristics of each transcript. Methods: Alternative splice isoforms of MYNN were identified using RNA sequencing (RNA-seq) and cloning techniques. Quantitative real-time polymerase chain reaction (qPCR) was employed to detect expression patterns in 11 tissues of Large White (LW) and Mashen (MS) pigs, and to study developmental expression patterns in cerebellum (CE), stomach (ST), and longissimus dorsi (LD). Results: The results showed that MYNN had two alternatively spliced isoforms, MYNN-1 (GenBank accession number: KY470829) and MYNN-2 (GenBank accession number: KY670835). MYNN-1 coding sequence (CDS) is composed of 1,830 bp encoding 609 AA, whereas MYNN-2 CDS is composed of 1,746 bp encoding 581 AA. MYNN-2 was 84 bp less than MYNN-1 and lacked the sixth exon. MYNN-2 was found to have one $C_2H_2$ type zinc finger protein domain less than MYNN-1. Two variants were ubiquitously expressed in all pig tissues, and there were significant differences in expression of different tissues (p<0.05; p<0.01). The expression of MYNN-1 was significantly higher than that of MYNN-2 in almost tissues (p<0.05; p<0.01), which testified that MYNN-1 is the main variant. The expression of two isoforms decreased gradually with increase of age in ST and CE of MS pig, whereas increased gradually in LW pig. In LD, the expression of two isoforms increased first and then decreased with increase of age in MS pig, and decreased gradually in LW pig. Conclusion: Two transcripts of pig MYNN were successfully cloned and MYNN-1 was main variant. MYNN was highly expressed in ST, CE, and LD, and their expression was regular. We speculated that MYNN plays important roles in digestion/absorption and skeletal muscle growth, whereas the specific mechanisms require further elucidation.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.627-632
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• 2007

FoxO1, FoxO3a and FoxO4 belong to the FoxO gene family, which play important roles in the PI3K/PKB pathway. In this study, we cloned the porcine FoxO1, FoxO3a and FoxO4 sequences and assigned them to SSC11p11-15, SSC1p13 and SSC xq13 using somatic cell hybrid panel (SCHP) and radiation hybrid panel (IMpRH). RT-PCR results showed that these three genes are expressed in multiple tissues. Sequencing of PCR products from different breeds identified a synonymous T/C polymorphism in exon 2 of FoxO3a. This FoxO3a single nucleotide polymorphism (SNP) can be detected by AvaII restriction enzyme. The allele frequencies of this SNP were investigated in Dahuabai, Meishan, Tongcheng, Yushan, Large White, and Duroc pigs. Association of the genotypes with growth and carcass traits showed that different genotypes of FoxO3a were associated with carcass length and backfat thickness between 6th and 7th ribs (BTR) and drip loss (p<0.05).