含P301L突变的Tau转基因小鼠纯合子品系的建立及鉴定

目的:建立含P301L突变的tau转基因小鼠的纯合子品系。方法:雄原核显微注射法获得含P301L突变的tau转基因阳性首建鼠,通过SYBR Green实时荧光定量PCR法和传统育种方式结合鉴定纯合子和杂合子。结果:共选育出95只纯合子,鉴定出的纯合子具有优于杂合子模拟老年痴呆生物学特性改变的优势。结论:外源性基因tau能稳定遗传,采用的SYBR Green实时荧光定量PCR和传统育种方式结合筛选鉴定纯合子和杂合子快速、经济、可靠。     

利用荧光定量PCR分析c-Ha-ras基因在C57-ras转基因小鼠中的拷贝数

目的:利用real-time PCR建立检测C57-ras转基因小鼠中外源c-Ha-ras基因拷贝数的简便方法,为药物安全性评价C57-ras转基因小鼠模型的繁殖和筛选提供数据支持。方法:以自建的人原癌基因c-Ha-ras转基因小鼠为研究对象,利用SYBR GreenⅠ荧光定量PCR的绝对定量法测定3个C57-ras转基因小鼠系的c-Ha-ras基因Ct值,通过与内参基因GAPDH比较计算获得转基因的拷贝数。结果:内参基因GAPDH的标准曲线为lg NGAPDH=-2.852Ct+26.236,外源基因c-Ha-ras的标准曲线为lg NRAS=-3.068Ct+39.186;经计算,NO.2、NO.3、NO.5系的F6代拷贝数分别为4、4和3,并且系内不同个体间拷贝数一致。结论:利用SYBR GreenⅠreal-time PCR技术建立了检测C57-ras转基因小鼠中外源基因拷贝数的方法,该方法操作简单,节约成本,为C57-ras致癌性模型的选留和应用提供了基础和技术手段,也为其他类似转基因品系中转基因拷贝数的确定提供了一种参考方法。     

利用实时荧光定量比较Ct法检测转基因小鼠外源基因拷贝数

目的：在建立转基因小鼠模型时,外源基因拷贝数是影响其表达水平和遗传稳定性的重要因素之一。外源基因拷贝数的精确测定,是建立转基因动物模型的重要环节。方法：合成cagA基因和内参基因GAPDH的引物,用标准曲线法测得cagA和GAPDH基因的扩增效率分别为97．6％和98．6％;将128拷贝阴性小鼠基因组和128拷贝c0鲥打靶质粒的混合物作为参照样品,取6只来自同一母本的F2阳性小鼠的128拷贝基因组作为待测样品;选取GAPDH作为内源参照基因,用比较Ct法对待测样品进行定量。结果：经计算,6只待测小鼠的cagA基因拷贝数平均值为8。结论：利用实时荧光定量PCR仪,呆用改良后的比较Ct法对转基因小鼠的外源基因拷贝数进行了精确测定。     

SYBR Green实时荧光定量PCR快速检测口腔幽门螺杆菌

目的建立一种快速、灵敏、特异的鉴定幽门螺杆菌实时荧光定量PCR方法。方法利用SYBR Green实时荧光定量PCR反应体系对口腔幽门螺杆菌进行检测。鉴定结果与临床常规鉴定方法相比较,评价其敏感度、特异度及重复性。结果通过48例样品的检测,结果显示实时荧光定量PCR法检测标本的鉴定结果与常规PCR鉴定方法的结果对比,特异度为100%,敏感度为100%;最小能检测到102个拷贝数的重组质粒;批内重复试验和批间重复试验结果均与常规鉴定方法结果相符。结论实时荧光定量PCR法鉴定口腔幽门螺杆菌,特异度和敏感度高,重复性好,且快速、简便。该方法有望成为检测口腔幽门螺杆菌感染的一种快速有效的方法。     

红系特异的GFP基因在转基因小鼠中的整合和表达

应用荧光定量PCR技术对由位点控制区LCR的HS2元件和 β 珠蛋白基因启动子指导的红系特异表达绿色荧光蛋白 (GFP)基因的转基因小鼠中外源基因拷贝数进行测定 ,使用荧光显微镜和流式细胞仪检测小鼠外周血中GFP的表达水平 ,并运用荧光原位杂交技术 (FISH)确定了其中两只转基因小鼠中外源基因的整合位点 ,结果表明 :在转基因小鼠中外源基因的拷贝数各不相同且相差较大 ,而且拷贝数与GFP基因的表达量之间未呈现出相关性 ;FISH分析确定出两只转基因小鼠的外源基因整合于不同的染色体上 ;杂交信号的强弱与拷贝数的多少相一致     

应用实时荧光PCR技术定性定量检测改良品质的转基因小麦

目的:对转入高分子量谷蛋白亚基的小麦中转基因成分进行实时荧光PCR定性定量检测。方法:针对转基因小麦品系中通用的ubiquitin启动子,NOS终止子以及标记基因bar基因进行定性筛选检测,同时用已知为单拷贝的Wx012基因作为小麦物种内源特异参照基因,用单粒B73转基因小麦提取基因组DNA建立内源基因和外源基因的标准曲线,对转基因小麦样品A进行定量检测,同时优化实时荧光PCR条件反应条件。定量检测结果为5.35%。结果:研究的实时荧光PCR技术对转基因小麦中转基因成分能够快速准确地进行定性定量检测。     

利用实时荧光定量PCR法检测转基因水稻外源基因拷贝数的研究

转基因植物中外源基因拷贝数是影响目的基因表达水平和遗传稳定性的重要因素，因此外源基因拷贝数的检测成为转基因研究的关键．利用高通量、快速、灵敏的SYBR Green Ⅰ荧光定量实时PCR法，检测了转大麦烟酰胺合成酶基因（NASl）水稻中外源基因拷贝数．以蔗糖磷酸合成酶基因（SPS）作为水稻的内源参照基因．通过梯度稀释法．分别获得了NAS1和SPS基因的Ct值与起始模板数的相关性标准曲线，相关系数分别为0．99976和0．99571，相关性高．通过目的基因NAS1和水稻内源参照基因SPS起始模板数的比较，获得了目的基因在转基因水稻中的拷贝数。在8株转基因株系中，1株为假阳性，1株拷贝数为1，3株拷贝数为2，其余3株拷贝数分别为3、4和7．而阴性对照拷贝数为0．这种方法快速、简便、准确，可以满足转基因育种工作中对后代优良株系的选择．     

转红色荧光蛋白基因唐鱼外源基因拷贝数的测定

目的:测定转红色荧光蛋白基因唐鱼的外源基因拷贝数。方法:以杂合F5代和杂合F6代转红色荧光蛋白基因唐鱼为材料,以其外源插入片段(pDsRed-mylz2)与基因组插入位点5′侧翼区之间的边界序列作为特异性内参片段,同时以外源整合的红色荧光表达载体序列(pDsRed-mylz2)作为目的基因,采用实时荧光定量PCR技术测定外源基因整合的拷贝数。结果:运用外源基因与特异内参在同批次实时荧光定量PCR中的初始拷贝数比值,得到杂合F5代和杂合F6代转红色荧光蛋白基因唐鱼中插入的外源红色荧光表达载体序列pDsRed-mylz2的拷贝数均值均为3。结论:转红色荧光蛋白基因唐鱼的外源基因拷贝数为3。     

实时荧光定量PCR的数据分析方法

实时荧光定量PCR是目前检测目的核酸拷贝数及分析靶基因在mRNA表达水平相对变化的主流技术。研究表明,分析结果的准确性依赖于数据分析方法的可靠性。我们简要综述实时荧光定量PCR的数据分析方法。     

Using real-time PCR to determine transgene copy number in wheat

Transgene copy number is usually determined by means of Southern blot analysis which can be time consuming and laborious. In this study, quantitative real-time PCR was developed to determine transgene copy number in transgenic wheat. A conserved wheat housekeeping gene,puroindoline-b, was used as an internal control to calculate transgene copy number. Estimated copy number in transgenic lines using real-time quantitative PCR was correlated with actual copy number based on Southern blot analysis. Real-time PCR can analyze hundreds of samples in a day, making it an efficient method for estimating copy number in transgenic wheat.     

Estimating transgene copy number in precocious trifoliate orange by TaqMan real-time PCR

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants, so transgene copy number analysis is identified as one most important task after obtaining transgenic plants. In this paper, TaqMan real-time PCR was used to estimate the copy number of exogenous MAC12.2 and NPTII genes in transgenic precocious trifoliate orange (Poncirus trifoliata [L.] Raf) in order to overcome the limitations of Southern blot analysis, which is labor-intensive, time-consuming, in considerable needs of DNA, etc. We developed a real-time PCR assay which permitted the determination of the copy number of transgene (MAC12.2 and NPTII), relative to a conserved endogenous gene (PtLTP) in transgenic lines. R value is 0.92 by comparing the results to that of Southern blot analysis, indicating a strong correlation coefficient between TaqMan real-time PCR assay and Southern blot method.     

Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR

In transgenic plants, transgene copy number can greatly influence the expression level and genetic stability of the target gene, making estimation of transgene copy number an important area of genetically modified (GM) crop research. Transgene copy numbers are currently estimated by Southern analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials and may involve hazardous radioisotopes. We report here the development of a sensitive, high-throughput real-time (RT)-PCR technique for estimating transgene copy number in GM rice. This system uses TaqMan quantitative RT-PCR and comparison to a novel rice endogenous reference gene coding for sucrose phosphate synthase (SPS) to determine the copy numbers of the exogenous beta-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes in transgenic rice. The copy numbers of the GUS and HPT in primary rice transformants (T0) were calculated by comparing quantitative PCR results of the GUS and HPT genes with those of the internal standard, SPS. With optimized PCR conditions, we achieved significantly accurate estimates of one, two, three and four transgene copies in the T0 transformants. Furthermore, our copy number estimations of both the GUS reporter gene and the HPT selective marker gene showed that rearrangements of the T-DNA occurred more frequently than is generally believed in transgenic rice.     

Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals

In this paper, we describe a rapid and accurate real-time quantitative PCR-based system to determine transgene copy number in transgenic animals. We used the 2(-deltadeltaCt) method to analyze different transgenic lines without the requirement of a control sample previously determined by Southern blot analysis. To determine the transgene copy number in several mouse lines carrying a goat beta-Lactoglobulin transgene, we developed a TaqMan assay in which a goat genomic DNA sample was used as a calibrator. Moreover, we used the glucagon gene as a reference control because this gene is highly conserved between species and amplifies with the same efficiency and sensitivity in goat as in mouse. With this assay, we provide an alternative simple method to determine the transgene copy number, avoiding the traditional and tedious blotting techniques. The assay's discrimination ability from our results is of at least six copies and, similar to the limitations of the blotting techniques, the accuracy of the quantification diminishes when the transgene copy number is high.     

Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum)

A TaqMan quantitative real-time PCR detection system was developed to examine transgene copy number in cotton. GhUBC1, a gene validated to be present as a single copy per haploid Gossypium hirsutum genome, was used as the endogenous reference to estimate copy number of GFP and selection marker NPTII in 28 T0 plants. This system was found to be more accurate than genomic Southern blot hybridization and could effectively tell homozygotes from heterozygotes in a T1 transgenic cotton population. Therefore it is suitable for efficient and cost effective early screening of transgenic seedlings and identifying transgene homozygotes in segregation populations.     

Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with<Emphasis Type="Italic">HMG I/Y</Emphasis> as an endogenous reference gene

In transgenic plants, the number of transgene copies can greatly influence the level of expression and genetic stability of the target gene. Transgene copy numbers are estimated by Southern blot analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials, and may involve hazardous radioisotopes. Here we report the development of a sensitive, convenient real-time PCR technique for estimating the number of transgene copies in transgenic rapeseed. This system uses TaqMan quantitative real-time PCR and comparison with a novel, confirmed single-copy endogenous reference gene, high-mobile-group protein I/Y (HMG I/Y), to determine the numbers of copies of exogenous β-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. TheGUS andnptII copy numbers in primary transformants (T₀) were calculated by comparing threshold cycle (C _T) values of theGUS andnptII genes with those of the internal standard,HMG I/Y. This method is more convenient and accurate than Southern blotting because the number of copies of the exogenous gene could be directly deduced by comparing itsC _T value to that of the single-copy endogenous gene in each sample. Unlike other similar procedures of real-time PCR assay, this method does not require identical amplification efficiencies between the PCR systems for target gene and endogenous reference gene, which can avoid the bias that may result from slight variations in amplification efficiencies between PCR systems of the target and endogenous reference genes.     

An improved method for determination of gene copy numbers in transgenic mice by serial dilution curves obtained by real-time quantitative PCR assay

Precise characterization of transgene insertion is necessary for phenotype interpretation of transgenic animals. To check for the presence of deletions, estimate the number of inserted transgene copies, and in addition, identify the zygosity of transgenic mice, gene copy numbers were determined by real-time quantitative PCR. Instead of correlating tested samples to a single relative standard curve, serial dilution curves were constructed for every mouse sample. A novel statistical approach was designed in which mice with the same copy number were characterized by the adjusted group mean and standard deviation common to the target sequence. This enabled us to characterize the variability of the obtained results, statistically compare different groups of mice and estimate precision and limits of the applied method.     

Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression

Bacterial artificial chromosomes (BACs) are excellent tools for manipulating large DNA fragments and, as a result, are increasingly utilized to engineer transgenic mice by pronuclear injection. The demand for BAC transgenic mice underscores the need for careful inspection of BAC integrity and fidelity following transgenesis, which may be crucial for interpreting transgene function. Thus, it is imperative that reliable methods for assessing these parameters are available. However, there are limited data regarding whether BAC transgenes routinely integrate in the mouse genome as intact molecules, how BAC transgenes behave as they are passed through the germline across successive generations, and how variation in BAC transgene copy number relates to transgene expression. To address these questions, we used TaqMan real-time PCR to estimate BAC transgene copy number in BAC transgenic embryos and lines. Here we demonstrate the reproducibility of copy number quantification with this method and describe the variation in copy number across independent transgenic lines. In addition, polymorphic marker analysis suggests that the majority of BAC transgenic lines contain intact molecules. Notably, all lines containing multiple BAC copies also contain all BAC-specific markers. Three of 23 founders analyzed contained BAC transgenes integrated into more than one genomic location. Finally, we show increased BAC transgene copy number correlates with increased BAC transgene expression. In sum, our efforts have provided a reliable method for assaying BAC transgene integrity and fidelity, and data that should be useful for researchers using BACs as transgenic vectors.     

An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.

Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL‐)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T‐DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL‐PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T₁ progeny from 26 T₀ plants showed that at least 19% of the lines carried multiple T‐DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T‐DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided.