实时荧光定量PCR法检测转基因小鼠拷贝数

目的利用实时荧光定量PCR法鉴定转基因小鼠外源基因插入拷贝数。方法以TG-CARK转基因首见鼠为研究对象,选取小鼠的高度保守基因Fabpi为内参,利用绝对定量的实时荧光PCR法鉴定转基因小鼠拷贝数,并与传统的Southern blot方法的定量结果进行比较。结果实时定量PCR鉴定的转基因拷贝数与Southernblot法完全一致,三只TG-CARK首见小鼠的拷贝数分别为1,7,45。结论实时定量PCR技术具有高准确性、高稳定性、高通量和低成本的优点,是比传统杂交技术更好的鉴定小鼠转基因拷贝数的方法。     

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.     

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.     

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。     

Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice.

Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms.     

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.     

Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKERS™-derived transgenic maize

. Quantitative real-time PCR (qRT-PCR) was adapted to estimate transgene copy number in transgenic maize callus and plants. WHISKERS™-derived transgenic callus lines and plants were generated using two different gene constructs. These transgenic materials represented a range of copy number. A 'standard curve' was established by mixing plasmid DNA with non-transgenic genomic maize DNA using a calculated ratio of target gene to host genome size. 'Estimated' copy number in the callus lines and plants using qRT-PCR was correlated with the 'actual' copy number based on Southern blot analysis. The results indicated that there was a significant correlation between the two methods with both gene constructs. Thus, qRT-PCR represents an efficient means of estimating copy number in transgenic maize.     

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.     

The methylation-free status of a housekeeping transgene is lost at high copy number

Transgenic mouse lines were established bearing tandem arrays of a fusion construct comprising the promoter region of a housekeeping gene, HMGCR, encoding 3-hydroxy 3-methylglutaryl CoA reductase, linked to a bacterial cat reporter gene encoding chloramphenicol acetyltransferase (CAT). CAT activity was observed in all transgenic mouse tissues examined. The methylation state of the fusion transgene was determined. In non-transgenic mice the endogenous HMGCR promoter is devoid of methylation while flanking regions are extensively modified. In HMGCR-cat transgenic mice the fusion gene promoter was found to be similarly hypomethylated. However, the extent of hypomethylation varied with copy number: methylation-free status was progressively lost with increasing transgene copy number. Further transgenic mouse lines were constructed carrying a truncated HMGCR regulatory region linked to cat. Transgene expression and hypomethylation were observed in testis but not in any other tissue, and testis-specific methylation-free status was also lost at high copy number. Loss of hypomethylation at high copy number may indicate that saturable DNA-binding factors normally protect the HMGCR promoter from methylation.     

Transgenic overexpression of cardiac actin in the mouse heart suggests coregulation of cardiac,skeletal and vascular actin expression

Previous studies have shown that depletion of cardiac actin by targeted disruption is associated with increased expression of alternative actins in the mouse heart. Here we have studied the effects of transgenic overexpression of cardiac actin using the -myosin heavy chain promoter. Lines carrying 7 or 8 copies of the transgene showed a 2-fold increase in cardiac actin mRNA and also displayed decreased expression of skeletal and vascular actin in their hearts. In contrast, a line with more than 250 copies of the transgene did not show a similar decrease in the expression of skeletal and vascular actin despite a 3-fold increase in cardiac actin mRNA. While the low copy number transgenic mice displayed hearts that were similar to non-transgenic controls, the high copy number transgenic line showed larger hearts with distinct atrial enlargement and cardiomyocyte hypertrophy. Further, while the low copy number transgenic mouse hearts were mildly hypocontractile when compared with non-transgenic mouse hearts, the high copy number transgenic mouse hearts were significantly so. We conclude that in the presence of a small number of copies of the cardiac actin transgene, homeostatic mechanisms involved in maintaining actin levels are active and negatively regulate skeletal and vascular actin levels in the heart in response to increased expression of cardiac actin. However, these putative mechanisms are either inoperative in the high copy number transgenic line or are countered by the enhanced expression of skeletal and vascular actin during cardiomyocyte hypertrophy.     

Transgene Expression Is Associated with Copy Number and Cytomegalovirus Promoter Methylation in Transgenic Pigs

Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, inheritance and expression instability of the transgene in transgenic animals is a major limitation. Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression. In the study, we generated two transgenic pigs by somatic cell nuclear transfer (SCNT) that express green fluorescent protein (GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and bisulfite sequencing were performed to determine transgene copy number and promoter methylation level. The correlation of transgene expression with copy number and promoter methylation was analyzed in individual development, fibroblast cells, various tissues, and offspring of the transgenic pigs. Our results demonstrate that transgene expression is associated with copy number and CMV promoter methylation in transgenic pigs.     

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.     

Stable Long Term Germ-Line Transmission of Transgene Integration Sites in Mice

Transgenic mice provide a valuable tool in all fields of basic and applied biological and medical research. In this study, we describe the fate of integrated transgenes in the mammalian host genome over a large number of generations. The stability of the germ-line transmission of integrated tyrosinase transgene copies was monitored up to generation F20 in a large number of individuals from seven transgenic mouse lines. Phenotypic and molecular genetic analysis of the offspring both within the different lines and in cross-breeding experiments revealed the high stability of the transgene integration sites in mice. Only very few individuals were affected by a transgene copy loss. These results indicate that, once homozygous transgenic lines are established, breeding programs can be continued to a high number of generations without further stringent molecular genetic analysis.     

Direct sequencing of PCR-amplified junction fragments from tandemly repeated transgenes.

When microinjected foreign genes integrate into the genomes of mice, multiple copies are frequently found clustered together at one location. How they concatamerize--by the integration of large linearized concatamers that are formed by simple end-to-end linkage, by circularization of individual DNA fragments and recombination, or by some other means--is not understood. In the transgenic animals studied thus far by ourselves and others, integration frequency and transgene copy number do not seem to be significantly influenced by the complementarity of the ends of the DNA fragments that have been microinjected. We have utilized PCR amplification and DNA sequence analysis to study selected transgene junctions at the nucleotide level. In two transgenic mice carrying the synthetic RSVcat gene (injected with noncomplementary overhangs on the fragment ends), ends were 'nibbled' from 1 to 62 bases before being joined to an adjacent gene copy. Repeated dinucleotides, providing the most minimal of homologies, are present in half of the characterized junctions. Determination of the relative copy number of the junctions in each mouse supports the idea that transgene complexes can undergo additional rearrangements after the initial formation event.     

人β_2m转基因小鼠的制备及鉴定(英文)

 制备人 β2m转基因小鼠 ,研究HLA B2 70 4基因的表达 .应用显微注射将人 β2m基因注入C5 7BL 6×昆明鼠和昆明鼠×昆明鼠F1代受精卵 .出生动物及其后代经PCR筛选 ,采用斑点杂交和Southern杂交对阳性鼠基因组DNA标本进行进一步鉴定和测定整合拷贝数 ,利用RT PCR检测阳性鼠中人 β2m转基因的表达 .6只原代仔鼠及 7只它们的下一代鼠 (F1)带有人 β2m基因 .由微注射基因后移卵出生的 86只小鼠中 ,C5 7BL 6×昆明鼠杂交仔鼠 35只 ,其中 4只阳性 (11 4 % ) ,昆明鼠×昆明鼠杂交仔鼠 5 1只 ,其中 2只阳性 (3 9% ) ,含有人 β2m基因的原代鼠×昆明鼠杂交仔鼠 2 0只 ,其中 7只阳性 .整合的转基因均为单拷贝 .Southern杂交证实上述阳性鼠确有转基因整合 .阳性鼠的皮肤、结肠、睾丸和脾脏组织中均有人β2m转基因mRNA的表达 .在转基因动物制备中 ,C5 7BL 6×昆明鼠F1代明显优于昆明鼠×昆明鼠F1代 .与人HLA B2 70 4基因相比 ,人 β2m基因不易整合 ,其整合率与整合拷贝数均较低 .得到的人 β2m转基因小鼠能够将人 β2m基困传给下一代 ,并可与人HLA B2 70 4转基因鼠交配 ,研究它的致病性     

Analysis of the human alpha globin upstream regulatory element (HS-40) in transgenic mice.

We have analysed the effect of a 1.4 kb segment of DNA containing the upstream alpha globin regulatory element (HS-40) on human alpha globin gene expression in fetal mice and lines of transgenic mice. High levels of tissue-specific, human alpha mRNA expression were seen in all transgenic animals and in this sense expression was position independent. However, the level of human alpha mRNA expression per integrated gene copy decreased during development and was inversely related to copy number. The limitation in expression with increasing gene copy number was shown to be in cis since homozygotes for the transgene produced twice as much human alpha mRNA as hemizygotes. In many respects HS -40 appears similar to single elements within the previously described beta globin locus control region and in cross breeding experiments we have shown that HS -40 behaves in a similar manner to such elements in transgenic mice.     

Accurate Determination of Zygosity in Transgenic Rice by Real-time PCR Does Not Require Standard Curves or Efficiency Correction

A number of quantitative, real-time PCR methods have been developed for determining transgene copy numbers in plants. Here, we demonstrate that the Roche LightCycler^TM system can be used to determine the zygosity of transgenic lines without the use of standard curves or efficiency correction calculations. We have developed a duplex PCR assay which permits the determination of zygosity, relative to a calibrator sample, in transgenic rice lines containing the gene for a viral glycoprotein. Our data demonstrate that unambiguous 2-fold discrimination of copy number can be attained by calculating relative copy number using the threshold crossing point (Ct) calculated by the LightCycler^TM software combined with delta delta Ct calculations, provided that the appropriate calibrator sample is included in each run. The method presented here is rapid, sensitive, robust and easy to optimise.     

Standard Addition Quantitative Real-Time PCR (SAQPCR): A Novel Approach for Determination of Transgene Copy Number Avoiding PCR Efficiency Estimation

Quantitative real-time polymerase chain reaction (qPCR) has been previously applied to estimate transgene copy number in transgenic plants. However, the results can be erroneous owing to inaccurate estimation of PCR efficiency. Here, a novel qPCR approach, named standard addition qPCR (SAQPCR), was devised to accurately determine transgene copy number without the necessity of obtaining PCR efficiency data. The procedures and the mathematical basis for the approach are described. A recombinant plasmid harboring both the internal reference gene and the integrated target gene was constructed to serve as the standard DNA. It was found that addition of suitable amounts of standard DNA to test samples did not affect PCR efficiency, and the guidance for selection of suitable cycle numbers for analysis was established. Samples from six individual T₀ tomato (Solanum lycopersicum) plants were analyzed by SAQPCR, and the results confirmed by Southern blot analysis. The approach produced accurate results and required only small amounts of plant tissue. It can be generally applied to analysis of different plants and transgenes. In addition, it can also be applied to zygosity analysis.     

Chromosomal localization and molecular organization of human genomic fragment containing TNF/LT locus in transgenic mice

Molecular organization, copy number and chromosomal localization of human TNF/LT locus fragment were determined in genomes of two transgenic mouse lines. Genome of the first one contains two copies, organized in head-to-tail manner and determined on eighth chromosome by karyotyping; single transgene copy of the second line is observed on the fifth chromosome. These mice could serve as valuable model for studying both human tumor necrosis factor and lymphotoxin physiological functions.