• Asian-Australasian Journal of Animal Sciences
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• 제30권8호
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• pp.1175-1182
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• 2017

Objective: To create genetically modified goat as a biopharming source of recombinant human lacotoferrin (hLF) with transcription activator-like effector nucleases. Methods: TALENs and targeting vector were transferred into cultured fibroblasts to insert hLF cDNA in the goat beta-lactoglobulin (BLG) locus with homology-directed repair. The gene targeted efficiency was checked using sequencing and TE7I assay. The bi-allelic gene targeted colonies were isolated and confirmed with polymerase chain reaction, and used as donor cells for somatic cell nuclear transfer (SCNT). Results: The targeted efficiency for BLG gene was approximately 10%. Among 12 Bi-allelic gene targeted colonies, five were used in first round SCNT and 4 recipients (23%) were confirmed pregnant at 30 d. In second round SCNT, 7 (53%), 4 (31%), and 3 (23%) recipients were confirmed to be pregnant by ultrasound on 30 d, 60 d, and 90 d. Conclusion: This finding signifies the combined use of TALENs and SCNT can generate biallelic knock-in fibroblasts that can be cloned in a fetus. Therefore, it might lay the foundation for transgenic hLF goat generation and possible use of their mammary gland as a bioreactor for large-scale production of recombinant hLF.

• Journal of Gastric Cancer
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• 제3권4호
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• pp.214-220
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• 2003

Purpose: The aim of this study was to investigate the frequency of loss of heterozygosity and the microsatellite instability at multiple tumor suppressor gene loci in gastric adenocarcinomas. Materials and Methods: Loss of heterozygosity and the microsatellite instability at several tumor suppressor gene loci were analyzed in 29 primary gastric carcinomas by using microdissection and the polymerase chain reaction. Results: Twenty-three ($79\%$) of the 29 cases demonstrated loss of heterozygosity at one or more loci. The frequency of loss of heterozygosity at the p53 locus was the highest ($63\%$) and those at the VHL, APC, p16, Rb, MEN1, BRCA1, DPC4, 3p21, and 16p13 region were $41\%,\;36\%,\;19\%,\;29\%,\;33\%,\;26\%,\;21\%,\;32\%,\;and\;11\%$, respectively. Compared with histological type, loss of heterozygosity was more common in diffuse-type gastric cancer (P<0.01). Interestingly, 9 of 10 tumors with allelic deletion at the p53 locus showed loss of heterozygosity at other tumor suppressor gene loci. The microsatellite instability was also detected in 6 ($20\%$) of the 29 cases at one or more loci. Conclusion: These data suggest that frequent loss of heterozygosity and the microsatellite instability at multiple tumor suppressor genes might be required for the development and the progression of gastric carcinomas and that p53 allelic loss may be the most frequent event in the development of gastric carcinomas.

• Asian Pacific Journal of Cancer Prevention
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• 제15권22호
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• pp.10009-10013
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• 2014

Background: Vitamin D receptor (VDR) gene has been a subject of extensive pharmacogenetic research recently. Association studies between different types of cancers including prostate cancer (PCa) and VDR gene polymorphism have also been conducted. The objective of this study was to find possible associations between PCa and VDR gene polymorphisms in the Pakistani population. Materials and Methods: A total of 162 subjects, including prostate cancer patients and controls, were genotyped for Apa I, Taq I and Fok I polymorphisms in the VDR gene using allele specific PCR, PCR-RFLP and direct DNA sequencing. Allelic frequencies were tested for Hardy-Weinberg equilibrium and associations between the genetic markers and PCa were calculated using logistic regression. Results: Apa I CC genotype was found to have strongest association with PCa risk, and "A" genotype was found to have protective effect. Fok I and Taq I did not have appreciable levels of association with PCa, although Taq I "TC" heterozygotes seemed to have some protective effect. Similarly the "C" allele of Fok I also seemed to have protective effect. Conclusions: To our knowledge, this is the first report showing association between VDR gene polymorphisms and PCa in Pakistan. Our findings may be somewhat skewed because of small sample size and tendency of consanguineous marriages in Pakistani society; nevertheless, it shows the trend of association and protective effects of certain VDR gene polymorphisms against PCa.

• Asian Pacific Journal of Cancer Prevention
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• 제17권4호
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• pp.2265-2270
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• 2016

Background: Chronic myeloid leukemia (CML) is one of the most frequent hematopoietic malignancies in the elderly population; however, knowledge is limited regarding the genetic factors associated with increased risk for CML. Polymorphisms affecting microRNA (miRNA) biogenesis or mRNA:miRNA interactions are important risk factors in the development of different types of cancer. Thus, we carried out a case-control study to test the association with CML susceptibility of gene variants located in the miRNA machinery genes AGO1 (rs636832) and GEMIN4 (rs2740348), as well as in the miRNA binding sites of the genes BRCA1 (rs799917) and KRAS (rs61764370). Materials and Methods: We determined the genotype of 781 Mexican-Mestizo individuals (469 healthy subjects and 312 CML cases) for the four polymorphisms using TaqMan probes to test the association with CML susceptibility. Results: We found a borderline association of the minor homozygote genotype of the KRAS_rs61764370 polymorphism with an increased risk for CML susceptibility (P = 0.06). After gender stratification, this association was significant only for women (odds ratio [OR] = 13.41, P = 0.04). The distribution of the allelic and genotypic frequencies of the four studied SNPs was neither associated with advanced phases of CML nor treatment response. Conclusions: To the best of our knowledge, this study is the first to show a significant association of the KRAS_rs61764370 SNP with CML. To further determine such an association of with CML susceptibility, our results must be replicated in different ethnic groups.

• Genomics & Informatics
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• 제10권2호
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• pp.117-122
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• 2012

A sample size with sufficient statistical power is critical to the success of genetic association studies to detect causal genes of human complex diseases. Genome-wide association studies require much larger sample sizes to achieve an adequate statistical power. We estimated the statistical power with increasing numbers of markers analyzed and compared the sample sizes that were required in case-control studies and case-parent studies. We computed the effective sample size and statistical power using Genetic Power Calculator. An analysis using a larger number of markers requires a larger sample size. Testing a single-nucleotide polymorphism (SNP) marker requires 248 cases, while testing 500,000 SNPs and 1 million markers requires 1,206 cases and 1,255 cases, respectively, under the assumption of an odds ratio of 2, 5% disease prevalence, 5% minor allele frequency, complete linkage disequilibrium (LD), 1:1 case/control ratio, and a 5% error rate in an allelic test. Under a dominant model, a smaller sample size is required to achieve 80% power than other genetic models. We found that a much lower sample size was required with a strong effect size, common SNP, and increased LD. In addition, studying a common disease in a case-control study of a 1:4 case-control ratio is one way to achieve higher statistical power. We also found that case-parent studies require more samples than case-control studies. Although we have not covered all plausible cases in study design, the estimates of sample size and statistical power computed under various assumptions in this study may be useful to determine the sample size in designing a population-based genetic association study.

• 생물정신의학
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• 제14권1호
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• pp.55-60
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• 2007

Objectives : There have been many association studies of panic disorder. However, studies about the dopaminergic function in panic disorder have been few. This study was aimed to examine the possible association of dopamine transporter gene(DAT1) polymorphism and panic disorder in Korean population. Methods : Ninety-eight patients with panic disorder(43 male(46.9%), mean age $42.13{\pm}10.88$ years) and one hundred and thirteen comparison subjects(67 male(40.7%), mean age $33.14{\pm}8.55$ years) were tested for DAT1 polymorphism. Genotypes of DAT1 with variable number of tandem repeats(VNTR) were determined using polymerase chain reaction. The differences of allelic frequency and genotype frequency distribution between patient and the control group were tested with Fisher-Freeman-Halton test. Results : There was association between DAT1 polymorphism and panic disorder(allele : p<0.03, genotype : p<0.05). The frequency of 10/10 homozygotes of DAT1 was significantly higher in control group(${\chi}^2$=4.452, df=1, p=0.035). Conclusion : These results in our Korean samples suggest that DAT1 polymorphism might be associated with the vulnerability of panic disorder. Possible association of dopaminergic genes and panic disorder should be investigated with future studies using larger and different population.

• Asian-Australasian Journal of Animal Sciences
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• 제24권6호
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• pp.752-756
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• 2011

Polymorphism of the second exon of the caprine leukocyte antigen-DRB3 gene (CLA-$DRB3^*02$) was investigated in this study. The 285 bp PCR product of 258 individuals from 10 domestic goat breeds in Southwest China was digested with restriction endonucleases PstI and HaeIII and then genotyped. Three alleles and 4 restriction digestion profiles were distinguished by digestion of the PCR fragment by PstI, and 8 alleles and 13 genotypes by HaeIII. For HaeIII restriction enzyme sites, the Chi-square ($X^2$) test showed that all goat breeds in this study did not fit with the Hardy-Weinberg equilibrium (p<0.01 or p<0.05). The highly polymorphic nature of CLA-$DRB3^*02$ was demonstrated and the ranges of gene heterozygosity (He) and polymorphism information content (PIC) were 0.36-0.63 and 0.32-0.55, respectively. Clustering analysis showed that the 10 goat breeds clustered into two groups and Dazu Black goat had a close genetic relationship with Chengdu Grey, Jintang Black and Nanjiang Yellow goats.

• Asian-Australasian Journal of Animal Sciences
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• 제29권10호
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• pp.1500-1507
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• 2016

Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause "double-muscling" trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN) which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제8권2호
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• pp.119-122
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• 1995

A total of 6 tamaraws were observed for determining coat color and measured for body conformation. Banding pattern of 4 blood proteins and 2 isozymes were analysed from the peripheral blood of two tamaraws. The coat colour of younger tamaraws were observed to be brown and adult was found to be complete black. The body weight, body length, withers height, head length, head width, horn length and tail length of adult male and female tamaraws were: 236 kg and 208 kg; 113.5 cm and 112.5 cm; 19.5 cm and 20 cm; 30.0 cm and 28.5 cm and 36.0 cm and 35.5 cm, respectively. The electrophoretic banding pattern, distance migrated and the allelic frequencies obtained in tamaraws were similar compared with that reported in swamp buffaloes. Although the sample size was very small, results of this study could be an important source of informations based on which a breeding programme can be designed to exploit some of the economic characters of tamaraw.

• Asian-Australasian Journal of Animal Sciences
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• 제8권4호
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• pp.357-364
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• 1995

A biochemical genetic study on blood enzyme/protein systems in some breeds/crosses of sheep in Malaysia was carried out using horizontal starch gel electrophoresis. Blood samples were collected from 435 sheep, representing 8 breeds/crosses. These included 5 wool sheep breeds (Thai Longtail, wiltshire, Suffolk, Dorsimal and cMBLx), 1 hair sheep breed (Barbados Blackbelly) and 2 hybrids between wool sheep and hair sheep (Cameroon ${\times}$ Thai Longtail and Bali Bali ${\times}$ Malin). Twenty loci systems were examined. Of these, ten ($HB{\beta}$, ALB, TF, XP, CAT, DIA1, EsA, GPI, ME and NP) exhibited genetic variation whereas the other ten (AAT, CA, DIA2, ${\alpha}GLO$, ${\alpha}GLU$, LDH, MDH, PEP[leu-gly-gly], 6PGD and SOD) were monomorphic. The allelic frequencies which were obtained in 10 polymorphic markers are assessed and compared with the results obtained by previous workers. The estimations of inbreeding coefficient, intrabreed variation and breed relationships have been critically discussed and are used to reveal some important recommendations.