Effect of ARHI on lung cancer cell proliferation, apoptosis and invasion in vitro

The purposes of this study were to elucidate the effects of ARHI (aplysia ras homolog I) on several biological features of lung cancer cells, including growth, proliferation and invasion, to collect experimental evidence for the future biological treatment of human lung cancer. The eukaryotic expression vector, pcDNA3.1–ARHI, was constructed and transfected into the human lung cancer cell line SK-MES-1. The biological properties of the resulting ARHI-expressing lung cancer cell line were evaluated using methyl thiazolyl tetrazolium assay, flow cytometry, and a Transwell invasion assay. Additionally, the influence of ARHI on the gene expression levels of cyclin D1, p27^KIP1, death-associated protein kinase 1 (DAPK1), and matrix metalloproteinases1/2 (MMP-1/2) was determined. Compared to the non-transfected SK-MES-1 cells and the cells transfected with the empty pcDNA3.1 plasmid, the ARHI-transfected cells displayed significantly reduced growth rates and decreased viability (P < 0.05). The ARHI-transfected cells also displayed a significantly higher percentage of cells in G1 phase (P < 0.05) and a lower percentage of cells in S phase (P < 0.05); a higher percentage of apoptosis (P < 0.05); and finally, a notable reduction in the basement membrane-penetration rate in the Transwell invasion assay (P < 0.05). Furthermore, it was determined that ARHI is capable of inhibiting the expression of cyclin D1, MMP-1, and MMP-2; however, ARHI promotes the expression of both p27^KIP1 and DAPK1 in SK-MES-1 cells. In conclusion, overexpression of ARHI gene might be associated with the inhibition of lung cancer cell growth, proliferation and invasion, and the promotion of apoptosis.     

Cloning of the Xuhuai Goat PPARγ Gene and the Preparation of Transgenic Sheep

This study aimed to clone the peroxisome proliferator-activated receptor γ (PPARγ) gene of the Xuhuai goat and to make transgenic sheep using intratesticular injection, so as to improve the meat quality and flavor by increasing the intramuscular fat content. The coding sequence of the goat PPARγ gene was 1,428 bp, encoding 475 amino acids. Its similarity with other species was 81 (chicken), 89 (mouse), 92 (pig), 98 (cow), and 99% (sheep). The similarity of the corresponding amino acid sequences was 92.9, 97.3, 98.3, 99.6, and 99.8%, respectively. The signal peptide region of the PPARγ protein was not found in this study, demonstrating that the protein is not secreted. RT-PCR and western blot revealed that PPARγ was expressed in vitro, and the protein was localized in the cytoplasm. The PPARγ gene was expressed in F₁ transgenic sheep at both the mRNA and the protein levels; the positive ratio was 13.7%.     

Characterization and SNP Identification of Part of the Goat Melanophilin Gene

The melanophilin (MLPH) gene has been characterized as the candidate gene for dilute coat color in some species, but little is known about it in the goat. In this study, part of the genomic DNA sequence (19,289 bp) containing the whole coding region of the MLPH gene from goat, as well as from sheep, was determined. We found 16 exons and 15 introns; the coding region was 1767 bp distributed in 15 exons (2–16). In sheep, the length of part of the genomic DNA sequence was 16,988 bp, with 16 exons and 15 introns, and the coding region was 1833 bp, distributed in 15 exons (2–16). Dozens of SNPs as well as some noticeable motifs in the goat MLPH gene were found during the process of sequencing and polymorphism screening. Based on the SSR Tool, three simple sequence repeat motifs were detected in the goat and sheep DNA sequences. Compared with cattle, we found insertions of 4 amino acids in goats and 26 amino acids in sheep.     

绵羊胎儿成纤维细胞体外培养及转基因研究

目的用增强型绿色荧光蛋白(EGFP)基因转染体外培养绵羊胎儿成纤维细胞,探讨绿色荧光蛋白对绵羊胎儿成纤维细胞生物学特性的影响.方法体外分离培养绵羊胎儿成纤维细胞,经脂质体介导EGFP基因转染第一代成纤维细胞,G418筛选10～12*!d,挑选转基因单克隆细胞,传代培养,进行细胞形态观察、生长曲线以及染色体核型分析,并进行了培养细胞性别鉴定.结果整合有EGFP基因的绵羊胎儿成纤维细胞生物学行为与未转染外源基因的细胞无明显差别,根据荧光强度可直接反应外源基因的表达量.结论 EGFP基因作为体内报告基因可用于转基因细胞的研究,并将整合有EGFP基因的转基因细胞为克隆动物提供核供体奠定了基础.     

CDADC1在人视网膜色素上皮细胞中功能研究

目的：研究细胞增殖相关基因CDADC1在人视网膜色素上皮细胞ARPE19中的表达及对ARPE19细胞增殖的影响。方法：采用基因重组技术构建荧光表达载体pEGFP－C1－CDADC1和真核表达载体pcDNA3．1-myc-CDADC1,用脂质体转染法转染ARPE19细胞,观察GFP—CDADC1融合蛋白在ARPE19细胞的表达定位,流式细胞仪测定CDADC1转染后对ARPE19细胞生长周期、凋亡的影响。结果：FP—CDADC1融合蛋白亚细胞定位显示,CDADC1低表达于ARPE19细胞胞浆,高表达于细胞核;pcD—NA3．1-myc-CDADC1瞬时转染ARPE19细胞显示24小时细胞无明显改变,48小时后重组质粒转染组S期细胞占细胞总数的19．37％,pcDNA3．1-myc空载质粒转染组S期细胞占10．87％,而空白对照组S期细胞占3．33％,重组质粒转染组与两对照组之间的差异有统计学意义（P〈0．01）。结论：CDADC1在增生性玻璃体视网膜病变（PVR）发生和发展过程中可促进DNA的合成,引起细胞增生。     

Heterochromatin Protein 1 Deleted Chromo Domain Decreases Gene Silencing of Transgene in Mouse

The heterochromatin protein 1 (HP1) regulates epigenetic gene silencing by promoting and maintaining chromatin condensation. To decrease gene silencing, the chromo domain (CD) in the M31 (the main HP1 in mouse) was deleted by site-directed mutagenesis. Vector pcDNA3.1(+)/M31-DeltaCD, in which the M31-DeltaCD is driven by the CMV promoter, and vector pcDNA3.1(+)/P1A3-M31-DeltaCD, in which the M31-DeltaCD is driven by a goat ss-casein promoter were constructed. The former vector was transfected into a murine fibroblast cell line, which can express enhanced green fluorescent protein (EGFP). EGFP expression, which was determined by flow cytometric analysis, increased approximately 80% in the transfected cells. After injection of the latter vector into transgenic mouse mammary glands, which can express human clotting factor IX (hFIX), the hFIX expression level in the mouse milk increased approximately 40-60% and hFIX in one mouse milk was maintained at a high concentration for over 10 days.     

Comparing the Effect of Uniaxial Cyclic Mechanical Stimulation and Chemical Factors on Myogenin and Myh2 Expression in Mouse Embryonic and Bone Marrow Derived Mesenchymal Stem Cells

Background: Environmental factors affect stem cell differentiation. In addition to chemical factors, mechanical signals have been suggested to enhance myogenic differentiation of stem cells. Therefore, this study was undertaken to illustrate and compare the effect of chemical and mechanical stimuli on Myogenin (MyoG) and Myosin heavy chani 2 (Myh2) expression of mouse bone marrowderived mesenchymal stem cells (BMSCs) and embryonic stem cells (ESCs). Methods: After isolation and expansion of BMSCs and generation of embryoid bodies and spontaneous differentiation of ESCs, cells were examined in 4 groups: (1) control group: untreated cells; (2) chemical group: cells incubated in myogenic medium (5-azacythidine and horse serum for BMSCs, dimethyl sulfoxide (DMSO) and horse serum for ESCs) for 5 days; (3) mechanical group: cells exposed to uniaxial cyclic strain (8%, 1 Hz, 24 h) and (4) chemical + mechanical group: cells incubated in myogenic medium for 4 days and then exposed to uniaxial cyclic strain. Real-time PCR was used to examine the expression of MyoG and Myh2 as specific myogenic markers. Results: suggested that mechanical loading, as a single factor, could elevate MyoG and Myh2 expression. Combining chemical with mechanical factor increases expression and there was no significant difference in MyoG expression of ESCs- and MSCs-chemical + mechanical groups; however, Myh2 expression was significantly higher in ESCs-mechanical group than that in the same group of MSCs.     

Targeted editing of goat genome with modular-assembly zinc finger nucleases based on activity prediction by computational molecular modeling

Zinc finger nuclease (ZFN) technology can mediate targeted genome modification to produce transgenic animals in a high-efficient and biological-safe way. Modular assembly is a rapid, convenient and open-source method for the synthesis of ZFNs. However, this biotechnology is hampered by multistep construction, low-efficiency editing and off-target cleavage. Here we synthesized and tested six pairs of three- or four-finger ZFNs to target one site in goat beta-lactoglobulin (BLG, a dominant allergen in goat milk) gene. Homology modeling was applied to build the structure model of ZFNs to predict their editing activities targeting at goat BLG gene. Goat fibroblast cells were transfected with plasmids that encoded ZFN pairs, and genomic DNA was isolated 72 h later for genome editing efficiency assay. The results of editing efficiency assay demonstrated that ZFNs with optimal interaction modes can edit goat BLG gene more efficiently, whereas ZFNs with unexpected interaction modes showed lower activities in editing BLG gene. We concluded that modular-assembly ZFNs can provide a rapid, public-available, and easy-to-practice platform for transgenic animal research and molecular modeling would help as a useful tool for ZFNs activity prediction.     

An alternate protocol for establishment of primary caprine fetal myoblast cell culture: an in vitro model for muscle growth study

Cultured myoblasts have been used extensively as an in vitro model in understanding the underlying mechanisms of myogenesis. Various protocols for establishing a pure myoblast culture have been reported which involve the use of special procedures like flow cytometry and density gradient centrifugation. In goat, only a few protocols for establishing a myogenic cell culture are available and these protocols use adult muscle tissues which often does not yield sufficient numbers of precursor cells with adequate proliferative capacity. Considering the disadvantages of adult myoblasts, we are proposing an alternate protocol using caprine fetus which does not require any special procedures. In the present study, more than 90–95% fetal-derived cell populations had the typical spindle to polyhedral shape of myoblast cell and stained positive for desmin, hence confirming their myogenic origin. These cells attained the maximum confluency as early as 3–4 d against 3 wk by adult myoblasts indicating a better growth potential. Further, quantitative real-time PCR analysis revealed a higher expression (p?<?0.01) of myogenic regulatory factors (i.e., myogenic determination factor 1, myogenic factor 5, and myogenin) and myostatin (MSTN) in the fetal as compared to the adult myoblasts. Consequently, higher proliferation and differentiation ability along with higher abundance of myogenic markers and MSTN make the fetal myoblasts a better in vitro model.     

绿色荧光蛋白与Wee1 Hu融合基因的构建及其真核表达

 构建Wee1Hu与增强型绿色荧光蛋白 (GFP)融合基因表达载体 ,并对其在真核细胞的表达及生物学效应进行研究 .应用基因工程技术构建重组载体 ,脂质体转染胰岛 β细胞株 ,流式细胞仪和免疫沉淀 Western印迹检测融合蛋白的表达 ,共聚焦显微镜分析融合蛋白在活细胞内的分布 ,3 D结构模建分析其结构特点 ,并用MTT法检测其生物学活性 .结果显示融合基因在瞬时或稳定转染的真核细胞中均获表达 ,融合蛋白主要分布在胞核区 ,融合蛋白中Wee1Hu的空间构象与天然Wee1Hu完全相同 ,表达融合蛋白的胰岛β细胞可避免其被细胞毒T淋巴细胞 (CTL)杀伤 .结果表明GFP Wee1Hu融合蛋白中 ,可发绿色荧光的分子标签GFP未能影响Wee1Hu的结构及其生物学活性 ;Wee1Hu可通过调控细胞周期而阻断CTL介导的细胞凋亡     

Construction, characterization and expression of full length cDNA clone of sheep YAP1 gene

RT-PCR, 5′RACE, 3′RACE were used to clone sheep full length cDNA sequence of YAP1 (Yes-associated protein 1), eukaryotic expression plasmid and a mutant that cannot be phosphorylated at Ser42 was successfully constructed. The amino acid sequence analysis revealed that sheep YAP1 gene encoded water-soluble protein and its relative molecular weight and isoelectric point was 44,079.0 Da and 4.91, respectively. Sub-cellular localization of YAP1 was in the nucleus, it is hydrophilic non-transmembrane and non-secreted protein. YAP1 protein contained 33 phosphorylation sites, seven glycosylation sites and two WW domains. The secondary structure of YAP1 was mainly composed of random coil, while the tertiary structure of domain area showed a forniciform helix structure. YAP1 gene was expressed in different tissues, the highest expression was in kidney and the lowest was in hypothalamus. The CDS of sheep YAP1was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, cloned into pMD19-T simple vector by T/A ligation. YAP1 coding region was further sub-cloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR, restriction enzyme and sequencing were used to confirm the recombinant plasmid. The sheep full-length YAP1 cDNA sequence is 1712 in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine by PCR, restriction digestion and sequencing. The result showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, this will help for further studies on the YAP1 protein expression and its biological activities.     

一种原位示踪外源目的基因表达载体的构建

应用分子克隆技术 ,分别将增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)、内部核糖体进入位点 (internalribosomeentrysite,IRES)和编码H-ras基因C端 2 0个氨基酸的DNA(rasc2 0 )片段插入真核表达载体pcDNA3,构建真核重组表达载体并将其命名为pZX。通过脂质体介导将该载体转染人宫颈癌细胞系HeLa ,培养过夜后在荧光显微镜下观察绿色荧光蛋白在细胞内的分布 ,并与pEGFP-C3质粒DNA转染该细胞系进行比较。结果表明 ,转染pZX载体的实验组细胞膜发出绿色荧光 ,而对照组绿色荧光则均匀弥散于整个细胞中 ,工具性载体pZX已构建成功     

重组pEGFP—C2-p100-TSN点突变质粒构建及表达

目的将6个不同的p100-TSN．Mutants基因片段分别定向连入PEGFP—C2质粒中,使P100-TSN突变蛋白能够与绿色荧光蛋白在COS7细胞中融合表达,从而为进一步研究p100蛋白TSN结构域的功能奠定实验基础。方法利用EcoR I和Xho I双酶切方法从6个pcDNA3．1（＋）-p100-TSN．Mutants重组质粒中分别获得p100-TSN．Mutants的cDNA片段,将其连人pEGFP—C2质粒载体中,再将成功构建的6个pEGFP—C2-p100-TSN．Mutants质粒分别转染COS7细胞中,荧光显微镜下观察绿色荧光蛋白表达。结果①将重组质粒进行双酶切鉴定可见p100-TSN．Mutants的cDNA片段;②转染重组质粒后可观察到绿色荧光蛋白的表达。结论①6个pEGFP—C2-p100-TSN．Mutants重组质粒构建成功;②p100-TSN突变蛋白可与绿色荧光蛋白在COS7细胞中融合表达。     

Production of functional human CuZn-SOD and EC-SOD in bitransgenic cloned goat milk

Human copper/zinc superoxide dismutase (CuZn-SOD) and extracellular superoxide dismutase (EC-SOD) are two superoxide dismutases that scavenge reactive oxygen species (ROS). Their biological role of eliminating oxidative stress caused by excessive ROS levels in living organisms has been utilized in medical treatment, preventing skin photoaging and food preservation. In this study, we employed two sequences that encode human CuZn-SOD and EC-SOD, along with goat beta-casein 5′ and 3′ regulatory elements, to construct mammary gland-specific expression vectors. Bitransgenic goats were generated using somatic cell nuclear transfer (SCNT), which employed co-transfection to generate bitransgenic goat fetal fibroblast cells as donor cells, and the expression of human CuZn-SOD and EC-SOD and their biological activities were assayed in the milk. PCR and Southern blot analysis confirmed that the cloned goat harbors both hCuZn-SOD and hEC-SOD transgenes. rhCuZn-SOD and rhEC-SOD were expressed in the mammary glands of bitransgenic goat, as determined by western blotting. The expression levels were 100.14?±?5.09 mg/L for rhCuZn-SOD and 279.10?±?5.38 mg/L for rhEC-SOD, as determined using ELISA. A total superoxide dismutase assay with WST-8 indicates that the biological activity of rhCuZn-SOD and rhEC-SOD in goat milk is 1451?±?136 U/mL. The results indicate that two expression vectors can simultaneously transfect goat fetal fibroblast cells as donor cells to produce transgenic goats by SCNT, and the CuZn-SOD and EC-SOD proteins secreted in the mammary glands showed biological activity. The present study thus describes an initial step in the production of recombinant human SODs that may potentially be used for therapeutic purposes.     

Regulation of nuclear factor of activated T cells (NFAT) and downstream myogenic proteins during dehydration in the African clawed frog

Xenopus laevis, otherwise known as the African clawed frog, undergoes natural dehydration of up to 30% of its total body water during the dry season in sub-Saharan Africa. To survive under these conditions, a variety of physiological and biochemical changes take place in X. laevis. We were interested in understanding the role that the calcineurin-NFAT pathway plays during dehydration stress response in the skeletal muscles of X. laevis. Immunoblotting was performed to characterize the protein levels of NFATc1-4, calcium signalling proteins, in addition to myogenic proteins (MyoD, MyoG, myomaker). In addition, DNA–protein interaction ELISAs were used to assess the binding of NFATs to their consensus binding sequence, and to identify the effect of urea on NFAT-binding. Our results showed that NFATc1 and c4 protein levels decreased during dehydration, and there were no changes in NFATc2, c3, and calcium signalling proteins. However, MyoG and myomaker both showed increases in protein levels during dehydration, thus indicating that the late myogenic program involving myoblast differentiation, but not satellite cell activation and myoblast proliferation, could be involved in preserving the skeletal muscle of X. laevis during dehydration. In addition, we observed that urea seems to reduce NFATc3-binding to DNA during control, but not during dehydration, possibly indicating that NFATc3 is protected from the denaturing effects of urea as it accumulates during dehydration. These findings expand upon our knowledge of adaptive responses to dehydration, and they identify specific protein targets that could be used to protect the skeletal muscle from damage during stress.