Oocyte-specific gene knockdown by intronic artificial microRNAs driven by Zp3 transcription in mice

Conditional knockout technology is a powerful tool for investigating the spatiotemporal functions of target genes. However, generation of conditional knockout mice involves complicated breeding programs and considerable time. A recent study has shown that artificially designed microRNAs (amiRNAs), inserted into an intron of the constitutively expressed gene, induce knockdown of the targeted gene in mice, thus creating a simpler method to analyze the functions of target genes in oocytes. Here, to establish an oocyte-specific knockdown system, amiRNA sequences against enhanced green fluorescent protein (EGFP) were knocked into the intronic sites of the Zp3 gene. Knock-in mice were then bred with EGFP transgenic mice. Our results showed that Zp3-derived amiRNA successfully reduced EGFP fluorescence in the oocytes in a size-dependent manner. Importantly, knockdown of EGFP did not occur in somatic cells. Thus, we present our knockdown system as a tool for screening gene functions in mouse oocytes.     

绵羊miR-127/FOXO4反馈环路及其对卵泡颗粒细胞凋亡相关基因表达的影响

旨在研究oar-miR-127/FOXO4反馈环路及其对绵羊卵泡颗粒细胞的作用。本研究利用Promoter 2.0预测绵羊miR-127启动子,利用JASPAR数据库预测转录因子FOXO4与oar-miR-127启动子区的结合位点,构建包含预测结合位点的pGL3-basic-miR-127荧光素酶报告载体和pcDNA-FOXO4过表达载体;利用TargetScan软件在线预测oar-miR-127与FOXO4基因3'-UTR区结合位点,构建包含预测结合位点的pmir-GLO-FOXO4野生型、突变型和缺失型荧光素酶报告基因重组质粒;将pGL3-basic-miR-127荧光素酶报告载体和pcDNA-FOXO4过表达载体共（或单独）转染体外培养的绵羊卵泡颗粒细胞,FOXO4突变型、野生型和缺失型重组质粒与miR-127 mimic/mimic NC共转染体外培养的293T细胞,48 h后检测荧光素酶活性;pcDNA-FOXO4过表达载体（miR-127 mimic/mimic NC）转染绵羊卵泡颗粒细胞48 h,利用qRT-PCR检测miR-127（FOXO4）及凋亡基因（Casp3、Bax及BCL2）的表达水平。过表达转录因子FOXO4显著降低了oar-miR-127启动子相对荧光素酶活性（P<0.05）和oar-miR-127表达水平（P<0.05）;miR-127 mimic和FOXO4野生型重组质粒共转染的颗粒细胞荧光素酶活性显著低于共转染miR-127 mimic和FOXO4缺失/突变型重组质粒的细胞（P<0.05）。转染miR-127 mimic的颗粒细胞中FOXO4表达量显著降低（P<0.05）,Casp3和Bax基因表达水平极显著升高（P<0.001）;过表达转录因子FOXO4的颗粒细胞中Casp3和Bax的表达水平无显著变化（P>0.05）,但BCL2相对表达量显著降低（P<0.05）。本研究证实了oar-miR-127与靶基因FOXO4间存在反馈环路,并促进绵羊卵泡颗粒细胞的凋亡,为研究其在绵羊卵泡发育中的作用机制奠定了基础。     

柠条锦鸡儿CkNCED1基因启动子的克隆及表达分析

利用染色体步移(Genome walking)技术,克隆柠条锦鸡儿CkNCED1基因上游启动子序列。经顺式元件预测分析,该序列除基本的启动元件之外,还含有2个脱落酸诱导响应元件ABRE、此外还含有多个逆境相关的元件。将CkNCED1基因启动子区连接到pGWB533植物表达载体上,构建Promoter::GUS载体,GUS组织化学染色结果表明,CkNCED1基因在植物的叶、根、茎、花和角果的维管组织中均有表达,且在叶片中表达强度最高。以上研究确定了柠条锦鸡儿CkNCED1基因的表达部位和强度。进一步说明CkNCED1基因在ABA合成调控中发挥重要作用,为深入研究基因功能奠定基础。     

猪miR-23a靶向调控Smad3基因表达的研究

为了确定miR-23a是否靶向调控Smad3基因,试验利用NotⅠ和XhoⅠ酶构建包含Smad3-3′-UTR的野生型(psiCHECKTM-2-W-Smad3-3′-UTR)和突变型双荧光酶报告载体(psiCHECKTM-2-M-Smad3-3′-UTR),并在PK-15细胞中转染miR-23amimics、miR-23ainhibitor及其阴性对照,采用双荧光酶检测试剂盒检测荧光素酶活性,用实时荧光定量PCR和Western blotting法分别检测Smad3基因的mRNA和蛋白表达水平。结果表明,将含Smad3-3′-UTR的野生型和突变型双荧光酶报告载体与miR-23amimic共转染PK-15细胞,野生型报告质粒表达的荧光素酶活性显著低于其阴性对照组(P<0.05);转染miR-23amimics能显著下调Smad3基因mRNA及其蛋白表达水平(P<0.05);而转染miR-23ainhibitor组与miR-23ainhibitor阴性对照组相比,Smad3基因蛋白表达差异不显著(P>0.05)。综合上述结果可知,猪miR-23a可靶向作用于Smad3基因。     

家蚕丝素蛋白H链基因启动子区域的克隆及活性分析

家蚕(Bombyxmori)丝素蛋白H链基因(fib-H)具有在后部丝腺组织专一性、高效性表达的特点。为利用其时空调控机制,通过PCR扩增方法克隆fib-H启动子片段,并进行序列分析,进而构建由fib-H启动子控制报告基因DsRed的重组载体pSK-FH-DsRed-PolyA,通过家蚕BmN细胞和丝腺进行瞬时表达。结果表明:克隆的fib-H启动子序列具有典型的丝腺特异性表达启动子的特征,并可以驱动DsRed报告基因在BmN细胞和家蚕后部丝腺组织中瞬时表达。     

Resveratrol-induced mitochondrial synthesis and autophagy in oocytes derived from early antral follicles of aged cows

Mitochondrial numbers increase during oocyte growth. In this study, we collected oocytes and granulosa cell complexes (OGCs) from early antral follicles (EAFs) of aged cows (> 120 months of age) and examined the effects of resveratrol on mitochondrial generation, degradation, and quality in oocytes grown in vitro. We also examined the effects of resveratrol on gene expression of the granulosa cells. Resveratrol (20 µM) enhanced the expression of SIRT1 and induced autophagy in both granulosa cells and oocytes derived from aged cows. Culturing the OGCs with resveratrol increased mitochondrial DNA copy numbers in oocytes grown in vitro. Furthermore, resveratrol increased the ATP content in oocytes and improved the developmental ability of the oocytes to the blastocyst stage. Gene expression profiles in granulosa cells, as evaluated by next-generation sequencing technology, revealed that resveratrol enhanced the expression of EIF2-related genes but downregulated the expression of mammalian target of rapamycin (mTOR)-, inflammation-, and cholesterol homeostasis-related genes in granulosa cells. In conclusion, resveratrol affected both oocytes and granulosa cells derived from aged cows and improved the quality of oocytes grown in vitro through upregulation of mitochondrial biogenesis and degradation in growing oocytes and conditioning of granulosa cells.     

猪GV/MⅡ期卵母细胞miRNAs表达谱及Npm2基因相关miRNAs筛选

旨在探索体外成熟前后猪卵母细胞miRNAs表达谱，并筛选参与调控Npm2基因表达的miRNAs。本试验回收屠宰场猪卵巢，收集GV期卵母细胞，经体外成熟培养后获得MⅡ期卵母细胞，分别提取约130个GV期和MⅡ期卵母细胞总RNA进行Illumina HiSeqTM 2500测序，获取miRNA测序数据，每个处理重复3次，筛选差异表达microRNAs并进行靶基因GO和KEGG聚类分析。结果表明，本研究成功构建了GV期和MⅡ期猪卵母细胞miRNAs表达谱，GV期和MⅡ期卵母细胞差异表达miRNAs有95个，具有相似表达模式的miRNA聚为一类，对95个差异表达miRNAs进行靶基因预测，共得到3 967个靶基因，经GO和KEGG富集分析表明，这些靶基因被富集于5 194个GO条目和212个信号通路，其中参与卵母细胞减数分裂成熟的信号通路有2个。在差异表达miRNAs中筛选到5个与Npm2基因相关的miRNAs。采用qRT-PCR对其中2个miRNAs进行验证，证实其表达趋势与测序结果一致。本研究筛选了GV期和MⅡ期卵母细胞差异表达miRNAs，推测差异表达的miRNAs可能通过代谢、卵母细胞减数分裂相关通路、孕激素介导的卵母细胞成熟通路等途径在卵母细胞体外成熟过程中发挥作用，并筛选出调控Npm2基因表达的miRNAs，研究结果可为进一步阐明miRNA对Npm2基因的调控及其在卵母细胞成熟过程中的作用提供依据。     

Transgenic Pigs with Pancreas-specific Expression of Green Fluorescent Protein

The development and regeneration of the pancreas is of considerable interest because of the role of these processes in pancreatic diseases, such as diabetes. Here, we sought to develop a large animal model in which the pancreatic cell lineage could be tracked. The pancreatic and duodenal homeobox-1 (Pdx1) gene promoter was conjugated to Venus, a green fluorescent protein, and introduced into 370 in vitro-matured porcine oocytes by intracytoplasmic sperm injection-mediated gene transfer. These oocytes were transferred into four recipient gilts, all of which became pregnant. Three gilts were sacrificed at 47–65 days of gestation, and the fourth was allowed to farrow. Seven of 16 fetuses obtained were transgenic (Tg) and exhibited pancreas-specific green fluorescence. The fourth recipient gilt produced a litter of six piglets, two of which were Tg. The founder Tg offspring matured normally and produced healthy first-generation (G1) progeny. A postweaning autopsy of four 27-day-old G1 Tg piglets confirmed the pancreas-specific Venus expression. Immunostaining of the pancreatic tissue indicated the transgene was expressed in β-cells. Pancreatic islets from Tg pigs were transplanted under the renal capsules of NOD/SCID mice and expressed fluorescence up to one month after transplantation. Tg G1 pigs developed normally and had blood glucose levels within the normal range. Insulin levels before and after sexual maturity were within normal ranges, as were other blood biochemistry parameters, indicating that pancreatic function was normal. We conclude that Pdx1-Venus Tg pigs represent a large animal model suitable for research on pancreatic development/regeneration and diabetes.     

DNA methylation-dependent modulator of Gsg2/Haspin gene expression

The Gsg2 (Haspin) gene encodes a serine/threonine protein kinase and is predominantly expressed in haploid germ cells. In proliferating somatic cells, Gsg2 is shown to be expressed weakly but plays an essential role in mitosis. Although the Gsg2 minimal promoter recognized by the spermatogenic cell-specific nuclear factor(s) has been found, to date, the molecular mechanism that differentially controls Gsg2 expression levels in germ and somatic cells remains to be sufficiently clarified. In this study, we analyzed the DNA methylation status of the upstream region containing the Gsg2 promoter. We found a tissue-dependent and differentially methylated region (T-DMR) upstream (-641 to -517) of the authentic promoter that is hypomethylated in germ cells but hypermethylated in other somatic tissues. Profiling of Gsg2 expression and DNA methylation status at the T-DMR in spermatogenic cells indicated that the hypomethylation of the T-DMR is maintained during spermatogenesis. Using the reporter assay, we also demonstrated that DNA methylation at the T-DMR of Gsg2 reduced the promoter activity by 60-80%, but did not fully suppress it. Therefore, the T-DMR functions as a modulator in a DNA methylation-dependent manner. In conclusion, Gsg2 is under epigenetic control.     

Insertion and stable expression of Gaussia luciferase gene by the genome of bovine viral diarrhea virus

As a tool to address selected issues of virus biology, we constructed a recombinant cDNA clone of bovine viral diarrhea virus (BVDV) expressing Gaussia luciferase (Gluc) reporter gene. A full-length genomic cDNA clone of a non-cytopathic BVDV isolate was assembled by recombination in yeast Saccharomyces cerevisiae. The Gluc gene was inserted between the N^pro and Core protein coding regions by recombination. The cDNA transcribed in vitro was infectious upon transfection of MDBK cells, resulting in reporter gene expression and productive virus replication. The rescued viruses were stable for 15 passages in cell culture, maintaining the replication kinetics, focus size and morphology similar to those of the parental virus. Expression and correct processing of the reporter protein were also maintained, as demonstrated by Gluc activity. These results demonstrate that genes up to 555 bp are simply assembled by a single step in yeast recombination and are stably expressed by this cDNA clone.     

Expression of foreign genes in chicks by hydrodynamics-based naked plasmid transfer in vivo

The study of gene function in vivo is considered one of the top achievements of modern biology, inasmuch as it provides tools to study gene function in the context of the whole animal. In chickens, techniques of DNA-mediated gene transfer are less advanced than in other animal or livestock models, and remain a significant challenge. The study presented here is the first to show that a hydrodynamics-based gene-transfer technique, originally developed for naked DNA transfer in mice, can be applied to chickens. Rapid injection of naked plasmids containing expression cassettes into the jugular vein of 6- to 10-day-old chicks resulted in specific expression of the transgenes. A CMV promoter-driven luciferase reporter gene was expressed at significant levels in the liver during the first 3 days post-injection with lower levels also detected in the kidney. Significantly, all injected birds showed detectable levels of luciferase expression. Similarly, injection of a plasmid containing the secreted human coagulation factor IX (hFIX) gene under the control of human alpha-1-anti-trypsin promoter resulted in detectable levels of the hFIX in the plasma during the first 2 days post-injection. The method described herein has the potential for a quick and simple route for gain and loss-of function experiments in chicken liver and kidney, as well as for studying systemic effects of secreted proteins and hormones.     

In vitro analysis of expression vectors for DNA vaccination of horses: the effect of a Kozak sequence

One of the prerequisite for developing DNA vaccines for horses are vectors that are efficiently expressed in horse cells.We have analysed the ectopic expression of the human serum albumin gene in primary horse cells from different tissues. The vectors used are of pcDNA and pUC origin and include the cytomegalovirus (CMV) promoter. The pUC vectors contain CMV intron A whereas the pcDNA vectors do not.Insertion of intron A diminished the expression from the pcDNA vectors whereas insertion of a Kozak sequence upstream of the gene in two types of pUC vectors increased significantly the in vitro expression in primary horse cells derived from skin, lung, duodenum and kidney.We report for the first time the significance of full consensus Kozak sequences for protein expression in horse cells in vitro.     

Baculovirus-mediated gene expression in chicken primary cells

A recombinant baculovirus was constructed containing an expression cassette with a reporter gene, green fluorescent protein, directed by a constitutive mammalian promoter: a human cytomegalovirus immediate early promoter/enhancer (CMV-IE). High titer virus was prepared with ultracentrifugation. Efficient gene delivery and expression were observed in the virus-treated chicken primary culture, myoblast cells, and whole embryonic fibroblast cells. It was noticed that an addition of sodium butyrate (a selective histone deacetylase inhibitor) to viral transduction medium extremely enhanced the reporter-gene expression. However, there is no effect of presence of trichostatin A observed. To maximize the reporter-gene expression, the baculoviral infection condition was optimized with both cell types. Our approaches demonstrated that recombinant baculovirus could efficiently deliver its genome DNA into chicken primary cells and that CMV-IE, a mammalian-cell-active promoter, was functional in chicken primary cells and could direct a high level of gene expression. Clearly, the recombinant baculovirus provides an alternative means for foreign gene delivery into avian cells.