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。     

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 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.     

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.     

Anthocyanin expression in marker free transgenic wheat and triticale embryos

Wheat and triticale plants were transformed by bombardment of isolated scutella with a genetic construct consisting of the two anthocyanin biosynthesis regulatory genes, C1 and Bperu, each under the control of the Ltp1 embryo-specific promoter. Transgenic plants were obtained in the absence of selective pressure and selectable marker gene at a transformation frequency of 0.93% and 1.55% in triticale and wheat, respectively. Initial screening of T₀ lines was performed by polymerase chain reaction (PCR), and further confirmation of PCR positives was done using real-time PCR and by phenotypic observation. In this study, quantitative real-time PCR (qRT-PCR) was developed to determine the transgene copy number in transgenic wheat and triticale. A conserved wheat housekeeping gene, puroindoline-b, was used as an internal control to calculate the transgene copy number in wheat and the SYBR green detection method with a standard curve, constructed on the basis of serially diluted plasmid, was used to calculate the transgene copy in triticale. Estimated transgene copies varied from 3 to 8 in wheat and 4 to 7 in triticale lines. The presence of anthocyanin regulatory genes, promoter, and termination sequences was detected in six wheat lines and four triticale lines. However, anthocyanin-pigmented embryos were only observed visually in mature T₁ seeds of two transgenic wheat lines and a single triticale line. Multisite insertion and reorganization of transgenes was likely the explanation for the failure of expression for the anthocyanin genes in the remaining wheat and triticale transgenic lines.     

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.     

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.     

Transgenic cotton expressing synthesized scorpion insect toxin AaHIT gene confers enhanced resistance to cotton bollworm (Heliothis armigera) larvae

A synthetic scorpion Hector Insect Toxin (AaHIT) gene, under the control of a CaMV35S promoter, was cloned into cotton via Agrobacterium tumefaciens-mediated transformation. Southern blot analyses indicated that integration of the transgene varied from one to more than three estimated copies per genome; seven homozygous transgenic lines with one copy of the T-DNA insert were then selected by PCR and Southern blot analysis. AaHIT expression was from 0.02 to 0.43% of total soluble protein determined by western blot. These homozygous transgenic lines killed larvae of cotton bollworm (Heliothis armigera) by 44–98%. The AaHIT gene could used therefore an alternative to Bt toxin and proteinase inhibitor genes for producing transgenic cotton crops with effective control of bollworm.     

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.     

Genetic transformation of prickly-pear cactus (Opuntia ficus-indica) by Agrobacterium tumefaciens

A system for genetic transformation of an elite prickly pear cactus (Opuntia ficus-indica L., cultivar Villa Nueva) by Agrobacterium tumefaciens was developed. Beginning with direct bacterial infection by using a hypodermic syringe to the meristematic tissue termed areoles, transgenic plants were obtained by selection with 100 mg l⁻¹ kanamycin. Transient and stable GUS activities were monitored on kanamycin-resistant shoots and regenerated plants, respectively. Genetic transformation of regenerated plants growing under selection was demonstrated by PCR and Southern blot analysis; transgene copy number in the genome of transgenic plants ranged from two to six, while the transformation frequency obtained by the system reported here was of 3.2%. This method may be useful for routine transformation and introduction of several important genes in prickly pear cactus.     

Determination of Transgene Copy Number in Stably Transfected Mammalian Cells by PCR-Capillary Electrophoresis Assay

The quantitative determination of transgene copy number in stably transfected mammalian cells has been traditionally estimated by Southern blot analysis. Recently, other methods have become available for appraisal of gene copy number, such as real-time PCR. Herein we describe a new method based on a fluorescently labeled PCR, followed by capillary electrophoresis. We amplified our target gene (prothrombin) and the internal control originating from genomic DNA (18S rRNA) in the same PCR tube and calculated the mean peak height ratio of the target:control gene for every cell clone sample. With this approach we identified stably transfected cell clones bearing the same transgene copy number. The results of our assay were confirmed by real-time PCR. Our method proves to be fast, low-cost, and reproducible compared with traditionally used methods. This assay can be used as a rapid screening tool for the determination of gene copy number in gene expression experiments.     

REAL-TIME PCR方法测定转基因小麦中外源基因拷贝数

采用SYBR GreenⅠ real-time PCR方法检测7株转基因小麦中外源半夏凝集素基因的拷贝数。以小麦蜡质基因（wx012）作为内参基因,以未转基因小麦基因组DNA为内参基因标准品进行5倍梯度稀释得到内参基因CT值与起始模板量的相关性标准曲线：y=-0.2667x+6.98;以含半夏凝集素基因（pta）的质粒DNA为目的基的因标准品同样进行5倍梯度稀释,建立目的基因CT值与起始模板量的相关性标准曲线：y=-0.2118x+4.53。通过SYBR GreenⅠ real-time PCR分别获得每一样本中目的基因和内参基因的CT值,将CT值分别代入标准曲线计算该样本中内参基因和目的基因起始模板量,目的基因与内参基因起始模板量比值即是目的基因在该转基因植株中的拷贝数。计算结果为：单拷贝的有1株,2个拷贝1株,3拷贝和4拷贝的各有2株,其中有1株为假阳性植株。     

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.     

Rapid and accurate determination of zygosity in transgenic animals by real-time quantitative PCR

Successful identification of homozygous and heterozygous transgenic animals with currently available techniques demands tedious and time-consuming procedures with a high proportion of ambiguous results. Real-time PCR is a quantitative and extremely precise method with high throughput that could be applied to the analysis of large numbers of animals differing only by a factor of two in the amount of target sequences. We defined the technical conditions of real-time PCR to co-amplify a transgene and a reference gene using two fluorogenic probes and the comparative cycle threshold method. We applied these conditions to the analysis of zygosity in a line of transgenic rats. Real-time PCR allowed clear-cut identification of all transgenic animals analysed (n=45) as homozygous or heterozygous. Southern blot analysis of these animals using an internal quantitative control and PhosphorImager quantification showed ambiguous results in six of them and was concordant with real-time PCR in the rest. Mating of homozygous and heterozygous animals, as defined by real-time PCR, showed transgene transmission to the offspring following expected Mendelian laws. Real-time PCR allows rapid, precise, non-ambiguous and high throughput identification of zygosity in transgenic animals. This technique could be helpful in the establishment of breeding programs for transgenic colonies and in experiments in which gene dosage effects could have a functional impact.