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ABSTRACT: The selection and use of animals with blood group 0 in the process of transplanting pig organs or tissues into humans can positively contribute to the control of acute immune rejection due to differences in blood groups. Exon-specific PCRs for the porcine blood group A transferase gene against genomic DNA from either blood group A or 0 animals resulted in the amplification failure of the A0 blood group gene exon 8 from blood group 0 animals. To characterize the genetic abnormality in the genome of blood group 0 animals, we screened bacterial artificial chromosome (BAC) clones from a Korean native pig BAC library which had the blood group 0 allele, and carried out shotgun sequencing. The analysis showed that the 0 allele has a large deletion between exon 7 of the A0 blood group gene and the neighbouring SURF6. We also showed that the ABO blood group antigens in humans and the A0 blood group antigens in pigs are coded by mutations within the orthologous glycosyltransferase gene. In addition, we developed a multiplex genotyping method for the porcine A0 blood group gene.
Animal Genetics 06/2011; 42(3):325-8. · 2.40 Impact Factor
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ABSTRACT: In an attempt to enable comprehensive high-resolution genotyping of the swine leukocyte antigen (SLA) gene, we performed a systemic analysis of nucleotide polymorphisms at introns 1 and 2 and exon 2 from diverse alleles of SLA-DRB1 and DRB1 pseudogenes. We amplified and cloned 16 partial sequences of SLA-DRB1 and DRB2 introns 1 and 2 from different alleles, and analyzed them together with sequences of four reported SLA-DRB pseudogenes, DRB2, 3, 4, and 5. The results showed the presence of extreme nucleotide variations within introns 1 and 2 of SLA-DRB-related genes including substitutions and deletions. On the basis of these results, we developed a comprehensive genotyping method for SLA-DRB1 by genomic polymerase chain reaction (PCR) and subsequent direct sequencing. A total of 415 animals were genotyped and 67 allelic combinations from 18 DRB1 alleles were identified. Among them, two alleles, SLA-DRB1*kn04 and *kn05, were previously unreported. SLA-DRB1 genotyping results from this study combined with those of SLA-DQB1 from our previous study presented 10 SLA class II haplotypes, three of which were previously unreported. Population analysis using seven different pig breeds showed differences in the allele frequency of SLA-DRB1 among breeds. Our results should benefit biological experiments requiring sequence-level genotyping results of SLA-DRB1 and further study of the complete genetic diversity of SLA-DRB1 using field samples.
Tissue Antigens 04/2011; 77(6):572-83. · 2.59 Impact Factor
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ABSTRACT: To enable the efficient analysis of a highly polymorphic swine major histocompatibility complex (MHC) class II gene, swine leukocyte antigen (SLA)-DQB1, we developed a simple and comprehensive high-resolution genotyping protocol. To obtain sufficient sequence information to design a set of common genotyping primers for SLA-DQB1, we cloned SLA-DQB1 introns 1 and 2 from 11 alleles with official four-digit allelic designations and sequenced the regions directly surrounding the SLA-DQB1 exon 2. Significant intronic nucleotide variations, including several deletions, were identified. Based on 733-bp assembled genomic sequences including introns 1 and 2 and exon 2 from 11 different alleles, a primer set was identified that allowed the ubiquitous amplification and analysis of the complete SLA-DQB1 exon 2 sequence. We then developed a method to directly sequence the amplified polymerase chain reaction (PCR) products without further experimental steps. We especially focused on avoiding superimposed peaks, which arose from the presence of allelic deletions, in the sequencing electropherogram of SLA-DQB1 heterozygous animals. The genotyping accuracy was evaluated by comparing the results of genomic sequence-based typing (GSBT) with those of other available methods, including cDNA sequence-based typing (SBT), low-resolution PCR typing with sequence-specific primers, allelic segregation analysis, and heterozygote simulation typing. In all cases, the results were consistent between SLA-DQB1 GSBT and previously reported methods or expected results. We applied it to genotype 350 animals from seven pig breeds. The observed level of heterozygosity from our genotyping was ∼51%, reflecting that a large portion of the animals were inbred miniature pigs. Among the seven pig breeds tested, the allelic diversity of SLA-DQB1 was highest in Berkshire pigs. In conclusion, we have developed a simple and effective SLA-DQB1 GSBT method by combining simple genomic DNA PCR and direct sequencing. Our new method may aid in the study of SLA diversity and disease resistance and susceptibility.
Tissue Antigens 10/2010; 76(4):301-10. · 2.59 Impact Factor
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ABSTRACT: In this report, we describe a simple, rapid, efficient and inexpensive strategy for sequencing inserted DNAs from clones of cDNA or gDNA libraries. This strategy uses PCR products directly amplified from transformed bacterial colonies, with universal primers within the vector. The method can be applied for sequencing cDNA or gDNA libraries with up to 4 similar to 5 kb insert sizes, without overnight liquid culture or plasmid DNA preparation steps. We successfully used this method to analyze clones from full-length, enriched cDNA libraries. Although simple, following this strategy will significantly help researchers to avoid unnecessary steps in the analysis of a cDNA library.
Biotechnology and Bioprocess Engineering. 01/2010; 15(5):817-821.
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ABSTRACT: Cited By (since 1996): 4, Export Date: 8 January 2013, Source: Scopus, doi: 10.1016/j.oceaneng.2010.02.007, Language of Original Document: English, Correspondence Address: Choi, J.E.; Hyundai Heavy Industries, 1, Jeonha-dong, Dong-gu, Ulsan, 682-792, South Korea; email: jechoi@hhi.co.kr, References: Chao, K.Y., Numerical propulsion simulation for the KCS container ship (2005) Proceedings of CFD Workshop, , Tokyo, Japan;
Ocean Engineering. 01/2010; 37(7):549-566.
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ABSTRACT: Sequences from the clones of full-length enriched cDNA libraries serve as valuable resources for functional genomic studies. We have analysed 1970 high-quality chromatograms (Phred value >or= 30) that were obtained from sequencing the 5' ends of brainstem, liver, neocortex and spleen clones derived from full-length enriched cDNA libraries from Korean native pigs. In addition, 50,000 pig expressed sequence tag (EST) sequence trace files were obtained from Genbank and combined with our sequencing information to facilitate SNP identification in silico. The process generated 8118 contigs, of which 239 included minimum one sequence from Korean native pig and contained 678 putative coding single nucleotide polymorphisms (cSNPs). Of these, 33 putative cSNPs were randomly selected for confirmatory analysis and validated using 20 pigs from four different breeds (Duroc, Landrace, Yorkshire, Korean native pig). Of the 33 putative cSNPs, 20 were confirmed (61%), which was similar to the frequency reported in other studies. We also identified 15 new cSNPs from the validation process, which were not detected by our in silico analysis. Our study shows that analysing genetically diverse pig breeds including the Korean native pig could serve as a useful strategy for generating a large number of cSNPs.
Journal of Animal Breeding and Genetics 05/2009; 126(2):127-33. · 1.46 Impact Factor
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ABSTRACT: Retrovirus-mediated exogenous gene transfection of somatic cells is an efficient method to produce transgenic embryos by somatic cell nuclear transfer (SCNT). This study evaluated whether efficiency of transgenic embryos production, by SCNT using fibroblast cells transfected by retrovirus vector, is influenced by the introduced transgene and whether recloning could further improve its efficiency. Transgenic cloned embryos were produced by SCNT of porcine foetal fibroblast cells transfected by either LNbeta-Z or LNbeta-enhanced green fluorescent protein (EGFP) retrovirus vector and evaluated for their developmental ability in vitro. Blastomeres from four-cell stage porcine embryos, produced by SCNT of foetal fibroblast cells transfected with LNbeta-EGFP retroviral vector, were subsequently recloned into enucleated metaphase II oocytes and evaluated for changes in chromatin configuration, in vitro embryo development and gene expression. Analysis of results showed that cleavage and blastocyst rates of porcine SCNT embryos, using LacZ (53.6 +/- 6.4%; 12.0 +/- 5.7%) or EGFP (57.5 +/- 6.3%; 10.1 +/- 4.1%) transfected fibroblasts, did not differ (p > 0.05) from those of non-transfected controls (60.9 +/- 8.2%; 12.3 +/- 4.0%). Recloning of blastomeres did not further improve the in vitro development rate. Interestingly, the nuclei of blastomere underwent slower remodelling process than somatic cell nuclei. Both cloned and recloned embryos showed 100% transgene expression and there were no evidence of mosaicism. In conclusion, our data shows that the efficiency of transgenic cloned embryos production by SCNT of somatic cells transfected with replication-defective retrovirus vector is not influenced by the transgene introduction into donor cells and recloning of four-cell stage blastomere could not further improve its efficiency.
Reproduction in Domestic Animals 11/2008; 44(1):106-15. · 1.36 Impact Factor
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H J Chung,
J K Pak,
B K Kim,
Y K Lee,
S K Im,
H H Seong,
S J Uhm,
H T Lee,
K S Chung,
K S Min, J H Kim,
N Wakasugi,
W K Chang
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ABSTRACT: Pregnancy block from exposure to foreign male mouse pheromones is sensitive to both male and female mating strain, as well as the foreign male pheromone-producing strain. Incidence of pregnancy block by male pheromones in mice is different depending on the combination of females, stud males and stimulus males. BALB/cA females mated with BALB/cA males showed a 100% pregnancy block when exposed to males of the DDK strain (Chung et al., 1997). In contrast, BALB/cA females mated with males of dissimilar strain show high rates of pregnancy even if they are exposed to DDK males; this difference is thought to be due to the difference in viability of embryos (Chung et al., 1999). The present study investigated how development of BALB/cA and F1 embryos differ under the influence of pregnancy block stimuli. F1 embryos had significantly higher numbers of cells than did the BALB/cA embryos (P<0.05) at day 3 of pregnancy after exposure to DDK males or after bromocriptine (dopamine agonist, 4 mg kg(-1), i.p.) treatment. Histological observation after bromocriptine treatment revealed that: (i) on day 4 of pregnancy, BALB/cA embryos tended to form a large blastocoel, but showed abnormalities such as degeneration of primitive endoderm and depression of the outer trophoblast-distal endoderm layer at the periphery of the inner cell mass (ICM) or detachment of the ICM from the outer layer. In contrast, 60-70% of F1 embryos were normal late blastocysts and incipient egg cylinders, but 28-40% of early blastocysts were degenerating; and (ii) day 5 BALB/cA embryos were in the range from incipient egg cylinder with a large proamniotic cavity to ectoplacental cone only, but their proximal endoderm and trophoblast-distal endoderm layer were degenerating. In contrast, the F1 embryos were mostly at the egg cylinder stage and maintained normal structure except for occasional enlargement of the developing yolk sac cavity. These results indicate that the lining of the inner surface of trophoblast by distal endoderm layer may be more firmly established and that the inner environment for development of F1 embryos may be more effectively maintained, thereby making them more resistant to deleterious influences due to pregnancy block stimuli than are BALB/cA embryos.
Reproduction (Cambridge, England) 10/2003; 126(3):327-35. · 3.09 Impact Factor
