• 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.

• IMMUNE NETWORK
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• 제12권1호
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• pp.33-39
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• 2012

Background: Therapeutic approaches using monoclonal antibodies (mAbs) against complement regulatory proteins (CRPs:i.e.,CD46,CD55 and CD59) have been reported for adjuvant cancer therapy. In this study, we generated a recombinant 1E8 single-chain anti-CD59 antibody (scFv-Fc) and tested anti-cancer effect.by using complement dependent cytotoxicity (CDC). Methods: We isolated mRNA from 1E8 hybridoma cells and amplified the variable regions of the heavy chain (VH) and light chain (VL) genes using reversetranscriptase polymerase chain reaction (RT-PCR). Using a linker, the amplified sequences for the heavy and light chains were each connected to the sequence for a single polypeptide chain that was designed to be expressed. The VL and VH fragments were cloned into the pOptiVEC-TOPO vector that contained the human CH2-CH3 fragment. Then, 293T cells were transfected with the 1E8 single-chain Fv-Fc (scFv-Fc) constructs. CD59 expression was evaluated in the prostate cancer cell lines using flow cytometry. The enhancement of CDC effect by mouse 1E8 and 1E8 scFv-Fc were evaluated using a cytotoxicity assay. Results: The scFv-Fc constructs were expressed by the transfected 293T cells and secreted into the culture medium. The immunoreactivity of the secreted scFv-Fc construct was similar to that of the mouse 1E8 for CCRF-CEM cells. The molecular masses of 1E8 scFv-Fc were about 120 kDa and 55 kDa under reducing and non-reducing conditions, respectively. The DNA sequence of 1E8 scFv-Fc was obtained and presented. CD59 was highly expressed by the prostate cancer cell line. The recombinant 1E8 scFv-Fc mAb revealed significantly enhanced CDC effect similar with mouse 1E8 for prostate cancer cells. Conclusion: A 1E8 scFv-Fc construct for adjuvant cancer therapy was developed.

• Asian Pacific Journal of Cancer Prevention
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• 제16권10호
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• pp.4215-4218
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• 2015

Gastric cancer (GC) is one of the most common malignancies in the world. It is the first cause of cancer deaths in both sexes In Iranian population. Circulating insulin-like growth factor-one (IGF-1) levels have been associated for gastric cancer. IGF-1 protein has central roles involved in the regulation of epithelial cell growth, proliferation, transformation, apoptosis and metastasis. Single nucleotide polymorphism in IGF-1 regulatory elements may lead to alter in IGF-1expression level and GC susceptibility. The aim of this study was to investigate the influence of IGF-1 gene polymorphism (rs5742612) on risk of GC and clinicopathological features for the first time in Iranian population. In total, 241 subjects including 100 patients with GC and 141 healthy controls were recruited in our study. Genotypes were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay with DNA from peripheral blood. The polymorphism was statistically analyzed to investigate the relationship with the risk of GC and clinicopathological properties. Logistic regression analysis revealed that there was no significant association between rs5742612 and the risk of GC. In addition, no significant association between genotypes and clinicopathological features was observed (p value>0.05). The frequencies of the CC, CT, and TT genotypes were 97%, 3%, and 0%, respectively, among the cases, and 97.9%, 2.1%, and 0%, respectively, among the controls. CC genotype was more frequent in cases and controls. The frequencies of C and T alleles were 98.9% and 1.1% in controls and 98.5% and 1.5% in patient respectively. Our results provide the first evidence that this variant is rare in Iranian population and it may not be a powerful genetic predisposing biomarker for prediction GC clinicopathological features in an Iranian population.

• Asian Pacific Journal of Cancer Prevention
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• 제15권24호
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• pp.10937-10941
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• 2015

Pathogenesis of lung cancer is a complicated biological process including multiple genetic and epigenetic changes. Since cigarette smoking is confirmed as the most main risk factor of non-small cell lung cancer (NSCLC), the aim of this study was to determine whether tobacco exposure plays a role in gene methylation. Methylation of the RAR-${\beta}$ gene were detected using methylation-specific polymerase chain reaction in DNA from 167 newly diagnosed cases with NSCLC and corresponding 105 controls. A significant statistical association was found in the detection rate of the promoter methylation of RAR-${\beta}$ gene between NSCLC and controls ($x^2$=166.01; p<0.01), and hypermethylation of the RAR-${\beta}$ gene was significantly associated with smoking status (p=0.038, p<0.05). No relationship was found between RAR-${\beta}$ gene methylation and pathologic staging including clinical stage, cell type, gender and drinking (p>0.05), and the methylation of RAR-${\beta}$ gene rate of NSCLC was slightly higher in stages III+IV (80.0%) than in I+II (70.8%). Similar results were obtained for methylation of the RAR-${\beta}$ gene between squamous cell carcinoma (77.9%) and other cell type lung cancer (73.9%). These results showed that the frequency of methylation increased gradually with the development of clinical stage in smoking-associated lung cancer patients, and tobacco smoke may be play a potential role in RAR-${\beta}$ gene methylation in the early pathogenesis and process in lung cancer, particularly squamous cell carcinoma. Aberrant promoter methylation is considered to be a promising marker of previous carcinogen exposure and cancer risk.

• International Journal of Oral Biology
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• 제33권4호
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• pp.205-211
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• 2008

Gingival overgrowth can cause dental occlusion and seriously interfere with mastication, speech, and dental hygiene. It is observed in 25 to 81% of renal transplant patients treated with cyclosporine A (CsA). CsA-induced gingival overgrowth (CIGO) is caused by quantitative alteration of the extracellular matrix components, particularly collagen. However, the molecular mechanisms involved in the pathogenesis of CIGO remain poorly understood, despite intense clinical and laboratory investigations. The aim of the present work is to identify differentially expressed genes closely associated with CIGO. Human gingival fibroblasts were isolated by primary explant culture of gingival tissues from five healthy subjects (HGFs) and two patients with the CIGO (CIGO-HGFs). The proliferative activity of CsA-treated HGFs and CIGO-HGFs was examined using the MTT assay. The identification of differentially expressed genes in CsA-treated CIGO-HGF was performed by differential display reverse transcriptase-polymerase chain reaction (RT-PCR) followed by DNA sequencing. CsA significantly increased the proliferation of two HGFs and two CIGO-HGFs, whereas three HGFs were not affected. Seven genes, including the beta subunit of prolyl 4-hydroxylase (P4HB) and testican 1, were upregulated by CsA in a highly proliferative CIGO-HGF. The increased P4HB and testican-1 mRNA levels were confirmed in CsA-treated CIGO-HGFs by semiquantitative RT-PCR. Furthermore, CsA increased type I collagen mRNA levels and suppressed MMP-2 mRNA levels, which are regulated by P4HB and testican-1, respectively. These results suggest that CsA may induce gingival overgrowth through the upregulation of P4HB and testican-1, resulting in the accumulation of extracellular matrix components.

• 미생물학회지
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• 제31권4호
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• pp.267-273
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• 1993

Escherichia coli 의 superoxide 라디칼 유도발현성 유전자의 탐색을 위하여 먼저 superoxide 라디칼에 의하여 발현이 유도되는 프로모터를 탐색하였다. 이를 위햐여, 프로모터 탐색벡터인 pJAC4 를 이용하여 E. coli MG1655 균주로부터 프로모터 library 를 구성하였다. Ampicillin 농도구 배정판법을 이용하여 프로모터 세기가 다른 클론들을 구별하여 프로모터 세기가 약한 것부터 중간정도까지 383개의 클론을 선별하였다. 이들 프로모터 클론들의 superoxide 라디칼 유도발현성을 paraquat 를 첨가한 ampicilin 농도구배평판에서 조사하였다. 이중 3개의 클론이 paraquat 에 의하여 유도됨을 확인하였고, 유도배율은 1.4-4배 정도이었다. 이들 프로모터의 염기서열을 결정한 결과 기종의 database 에 등록되어 있는 E. coli 염기서열들과는 다른 새로운 유전자임을 알았다. 이들 프로모터에 대하여 primer 연장법과 SS1 뉴클리아제 분석을 총하여 전사개시 자리를 결정하였고, paraquat 처리시 mRNA 의 양이 증가함을 관찰하였다. 특히 클론5의 경우는 두개의 전사개시 위치를 가지고 있으며, paraquat 처리시 상위 부분의 전사개시자리가 성택적으로 사용됨을 알 수 있었다. 프로모터 서열을 검색한 결과, RNA 중합효소($E\sigma^{70}$)에 의햐여 인지되는 -10과 -35부위를 가진 클론은 클론 5 뿐이었고, 나머지는 보존된 염기서열을 찾을 수 없었다. 이는 이들 프로모터들이 독특한 부류에 속하는 종류들로서, 새로운 전사조절인자를 요구할 가능성을 시사한다.

• 생명과학회지
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• 제18권3호
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• pp.336-343
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• 2008

본 연구에서는 인체전립선 상피세포인 267B1 세포에서 HDAC 저해제인 TSA에 의한 증식억제가 apoptosis 유도에 의한 것임을 제시하였다. 이러한 TSA에 의한 267B1 세포의 apoptosis에는 c-IAP-1 및 c-IAP-2와 같은 IAP family의 발현감소가 동반되었으나 Bax 및 Bcl-2와 같은 Bcl-2 family의 발현에는 큰 변화가 없었다. 그리고 TSA에 의한 267Bl 세포의 apoptosis는 caspase의 활성에 의한 표적 단백질들의 분해와 연관성이 있었다. 또한 TSA에 의한 apoptosis 유도에서 $NF-{\kappa}B$의 활성이 증가된다는 것을 세포질에서 $NF-{\kappa}B$의 핵 내로의 이동에 따른 전사활성의 증가 현상에 의한 것임을 다양한 방법으로 제시하였다. 본 연구의 결과는 TSA와 같은 HDAC 저해제에 의한 apoptosis 유도에는 $NF-{\kappa}B$의 활성 증가가 동반될 수 있음을 보여주는 결과로서 HDAC 저해제의 항암활성에 대한 $NF-{\kappa}B$의 새로운 역할 가능성을 제시하여 주는 것으로서 이에 관한 추가적인 연구의 필요성을 제시하였다.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2006년도 추계학술대회 및 정기총회
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• pp.32-44
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• 2006

To investigate non-aflatoxin-production of A. oryzae at the molecular level, an aflatoxin biosynthesis gene homolog cluster of RIB 40 was analyzed. Although most genes in the corresponding cluster exhibited from 97 to 99 % similarity to those of Aspergillus flavus, three genes shared 93 % similarity or less. In addition, although slight expression of aflR, positive transcriptional regulator gene, was detected in some A. oryzae strains having seven aflatoxin biosynthesis homologous genes, other genes related to aflatoxin production were not detected. RIB strains were mainly divided into group 1, having seven aflatoxin biosynthesis homologous genes (aflT, nor-i, aflR, norA, avnA, verB, and vbs), and group 2, having three homologous (avnA, verB, and vbs). Partial aflatoxin homologous gene cluster of RIB62 from group 2 was sequenced and compared with that of RIB40 from group 1. RIB62 showed a large deletion upstream of ver-1 with more than half of the aflatoxin homologous gene cluster missing including aflR, a positive transcriptional regulatory gene. Adjacent to the deletion of the aflatoxin homologous gene cluster, RIB62 has a unique sequence of about 8kb and a telomere. Southern analysis of A. oryzae RIB strains with four kinds of probe derived from the unique sequence of RIB62 showed that all group 2 strains have identical hybridizing signals. Polymerase chain reaction with specific primer set designed to amplify the junction between ver-1 and the unique sequence of RIB62 resulted in the same size of DNA fragment only from group 2 strains. Based on these results, we developed a useful genetic tool that distinguishes A. oryzae group 2 strains from the other groups' strains and propose that it might have differentiated from the ancestral strains due to chromosomal breakage.

• Molecules and Cells
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• 제45권3호
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• pp.122-133
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• 2022

The aim of this study was to investigating whether lncRNA H19 promotes myocardial fibrosis by suppressing the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β axis. Patients with atrial fibrillation (AF) and healthy volunteers were included in the study, and their biochemical parameters were collected. In addition, pcDNA3.1-H19, si-H19, and miR-29a/b-3p mimic/inhibitor were transfected into cardiac fibroblasts (CFs), and proliferation of CFs was detected by MTT assay. Expression of H19 and miR-29a/b-3p were detected using real-time quantitative polymerase chain reaction, and expression of α-smooth muscle actin (α-SMA), collagen I, collagen II, matrix metalloproteinase-2 (MMP-2), and elastin were measured by western blot analysis. The dual luciferase reporter gene assay was carried out to detect the sponging relationship between H19 and miR-29a/b-3p in CFs. Compared with healthy volunteers, the level of plasma H19 was significantly elevated in patients with AF, while miR-29a-3p and miR-29b-3p were markedly depressed (P < 0.05). Serum expression of lncRNA H19 was negatively correlated with the expression of miR-29a-3p and miR-29b-3p among patients with AF (r_s = -0.337, r_s = -0.236). Moreover, up-regulation of H19 expression and down-regulation of miR-29a/b-3p expression facilitated proliferation and synthesis of extracellular matrix (ECM)-related proteins. SB431542 and si-VEGFA are able to reverse the promotion of miR-29a/b-3p on proliferation of CFs and ECM-related protein synthesis. The findings of the present study suggest that H19 promoted CF proliferation and collagen synthesis by suppressing the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β axis, and provide support for a potential new direction for the treatment of AF.

• 농업과학연구
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• 제25권2호
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• pp.216-224
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• 1998

본 연구는 축협중앙회 한우개량부에서 사육 중인 한우 암소 253두와 축산기술연구소에서 사육 중인 Holstein 113두의 혈액으로 부터 genomic DNA를 추출하고, PCR-RFLP기법에 의해 ${\beta}$-lactoglobulin과 ${\kappa}$-casein 유전자좌위의 유전적 다형을 분석하여 한우와 유우 집단에 대한 이들 유전자의 유전적 구조를 분석함으로써 한우와 유우 개량을 위한 기초 및 응용자료를 제공하고자 실시하였던 바 얻어진 결과를 요약하면 다음과 같다. 1. 한우와 Holstein종의 genomic DNA로 부터 PCR기법을 이용하여 ${\beta}$-lactoglobulin 과 ${\kappa}$-casem의 유전자좌를 증폭한 결과 각각 530bp와 262bp의 단편이 증폭되었음을 확인하였다. 2. ${\beta}$-lactoglobulin 증폭 산물에 대한 Hae III 제한효소의 처리결과, ${\beta}$-lactoglobulin AA형은 153bp와 109bp의 단편을, AB형은 153bp, 109bp, 79bp 및 74bp의 단편을, 그리고 BB형은 109bp, 79bp 및 74bp의 단편을 나타내었다. 3. ${\kappa}$-casein 유전자좌의 증폭산물에 대한 Taq I의 제한효소 처리결과, ${\kappa}$-casein AA형은 530bp의 단편을, AB형은 530bp, 344bp 및 186b의 단편을, 그리고 BB형은 344bp 및 186mp의 단편을 나타내었다. 4. ${\beta}$-lactoglobulin의 유전자형 및 유전자 빈도에 있어서, 한우의 ${\beta}$-lactoglobulin AA, AB 및 BB 유전자형 의 빈도는 각각 6.72, 26.09 및 67.19%이었으며, ${\beta}$-lactoglobulin A 및 B 유전자 빈도는 각각 0.197 및 0.803이었고, Holstein종의 ${\beta}$-lactoglobulin AA, AB 및 BB 유전자형 빈도는 각각 35.40, 56.64 및 7.96%이었으며, ${\beta}$-lactoglobulin A 및 B 유전자 빈도는 각각 0.637 및 0.363이었다. 5. ${\kappa}$-casein의 유전자형 및 유전자 빈도에 있어서, 한우의 ${\kappa}$-casein AA, AB 및 BB 유전자형의 빈도는 각각 46.25, 39.13 및 14.62%이었으며, K-casein A 및 B 유전자 빈도는 각각 0.658 및 0.342이었고, Holstein종의 ${\kappa}$-casein AA, AB 및 BB유전자형 빈도는 각각 60.18, 38.94 및 0.88%이었고, ${\kappa}$-casein A 및 B 유전자 빈도는 각각 0.796 및 0.204 이었다. 6. 이상의 결과로써 ${\beta}$-lactoglobulin과 ${\kappa}$-casein의 유전자 빈도는 한우에서 ${\beta}$-lactoglobulin A 및 B 대립 유전자 빈도는 각각 0.197 및 0.803이었고 ${\kappa}$-casein A 및 B 대립유전자 빈도는 각각 0.658 및 0.342이었다. 그러나 Holstein 종에서는 ${\beta}$-lactoglobulin A 및 B 대립 유전자 빈도는 0.637 및 0.363이었고, ${\kappa}$-casein A 및 B 대립유전자 빈도는 각각 0.796 및 0.204이었다.