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 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.     

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.     

Spontaneous germline amplification and translocation of a transgene array.

The majority of the mammalian genome is thought to be relatively stable throughout and between generations. There are no developmentally programmed gene amplifications as seen in lower eukaryotes and prokaryotes, however a number of unscheduled gene amplifications have been documented. Apart from expansion of trinucleotide repeats and minisatellite DNA, which involve small DNA elements, other cases of gene or DNA amplifications in mammalian systems have been reported in tumor samples or permanent cell lines. The mechanisms underlying these amplifications remain unknown. Here, we report a spontaneous transgene amplification through the male germline which resulted in silencing of transgene expression. During routine screening one mouse, phenotypically negative for transgene expression, was found to have a transgene copy number much greater than that of the transgenic parent. Analysis of the transgene expansion revealed that the amplification in the new high copy transgenic line resulted in a copy number approximately 40-60 times the primary transgenic line copy number of 5-8 copies per haploid genome. Genetic breeding analysis suggested that this amplification was the result of insertion at only one integration site, that it was stable for at least two generations and that the site of insertion was different from the site at which the original 5-8 copy array had integrated. FISH analysis revealed that the new high copy array was on chromosome 7 F3/4 whereas the original low copy transgene array had been localised to chromosome 3E3. DNA methylation analysis revealed that the high copy transgene array was heavily methylated. The amplification of transgenes, although a rare event, may give insight into amplification of endogenous genes which can be associated with human disease.     

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。     

Comparison of Biolistic and Agrobacterium -mediated Transformation Methods on Transgene Copy Number and Rearrangement Frequency in Rice

Transferring foreign DNA into plant cells by biolistic and Agrobacterium -mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG-CAM, into rice (BX)Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium -mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium -mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two-cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium -mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium -mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium -mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangement in transgenic plants transformed with the same plasmid by two different transformation methods.     

Hypersensitive site 4 of the human beta globin locus control region.

The Locus Control Region (LCR) of the human beta globin gene domain is defined by four erythroid-specific DNasel hypersensitive sites (HSS) located upstream of this multigene cluster. The LCR confers copy number dependent high levels of erythroid specific expression to a linked transgene, independent of the site of integration. To assess the role of the individual hypersensitive sites of the LCR, we have localized HSS4 to a 280bp fragment that is functional both in murine erythroleukaemia (MEL) cells and in transgenic mice. This fragment coincides with the major area of hypersensitivity 'in vivo' and contains a number of DNasel footprints. Bandshift analysis shows that these footprints correspond to binding sites for the erythroid specific proteins GATA1 and NF-E2 and a number of ubiquitous proteins, including jun/fos, Sp1 and TEF2.     

Detection of differential gene copy number using denaturing high performance liquid chromatography

Genomic rearrangements leading to deletion or duplication of gene(s) resulting in alterations in gene copy number underlie the molecular lesion in several genetic disorders. Methods currently used to determine gene copy number including real time PCR, southern hybridization, fluorescence in situ hybridization, densitometric scanning of PCR product etc. have certain disadvantages and are also expensive and time consuming. Herein, we describe a simple and rapid method to assess gene copy number using denaturing high performance liquid chromatography (dHPLC). We used X chromosome genes as model to compare the gene copy numbers present on this chromosome in males and females. DNA from these samples were amplified by biplex PCR using primer pairs specific for X chromosome genes only (target gene) and for genes present on both X and Y chromosomes (internal control). Amplified products were analyzed using HPLC under non-denaturing conditions. The ratio of peak areas (target gene/internal control) of the amplified products was approximately twice in female samples than male samples (p < 0.001) demonstrating that the differential gene copy number can be easily detected using this method. This method can potentially be used for diagnostic purpose where the need is to distinguish samples based on the differential gene copy numbers.     

SV40 intron,a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells have become the most widely utilized mammalian cell line for the production of recombinant proteins. However, the product yield and transgene instability need to be further increased and solved. In this study, we investigated the effect of five different introns on transgene expression in CHO cells. hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, Chinese hamster EF‐1alpha gene intron 1 and intervening sequence intron were cloned downstream of the eGFP expression cassette in a eukaryotic vector, which was then transfected into CHO cells. qRT‐PCR and flow cytometry were used to explore eGFP expression levels. And gene copy number was also detected by qPCR, respectively. Furthermore, the erythropoietin (EPO) protein was used to test the selected more strong intron. The results showed that SV40 intron exhibited the highest transgene expression level among the five compared intron elements under transient and stable transfections. In addition, the SV40 intron element can increase the ratio of positive colonies and decrease the coefficient of variation in transgene expression level. Moreover, the transgene expression level was not related to the gene copy number in stable transfected CHO cells. Also, the SV40 intron induced higher level of EPO expression than IVS intron in transfected CHO cell. In conclusion, SV40 intron is a potent strong intron element that increases transgene expression, which can readily be used to more efficient transgenic protein production in CHO cells.     

Structure and function of the murine beta-globin locus control region 5' HS-3.

We previously identified the murine homologue of the human beta-globin Locus Control Region (LCR) 5' HS-2. The lambda clone containing murine 5' HS-2 extends approximately 12 kb upstream from this site; here, we report the sequence of this entire upstream region. The murine homologue of 5' HS-3 is located approximately 16.0 kb upstream from the mouse epsilon y-globin gene, but no region homologous to human 5' HS-4 was present in our clone. Using a reporter system consisting of a human gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo), we tested murine LCR fragments extending from -21 to -9 kb (with respect to the epsilon y-globin gene cap site) for activity in classical enhancer and integration site assays in K562 and MEL cells. 5' HS-2 behaved as a powerful enhancer and increased the number of productive integration events (as measured by a colony assay) in both K562 and MEL cells. 5' HS-3 had no activity in K562 cells or in transiently transfected MEL cells, but was nearly as active as 5' HS-2 in the MEL cell colony assay. Two additional tests confirmed the identification of murine 5' HS-3: first, a DNA fragment containing 5' HS-3 confers copy number-dependent, integration-site independent inducibility on a linked beta-globin gene in the MEL cell environment. Secondly, a strong DNAseI hypersensitive site maps to the location of the 5' HS-3 functional core in chromatin derived from MEL cells. Collectively, these data suggest that we have identified the murine homologue of human 5' HS-3, and that this site is functional when integrated into the chromatin of MEL cells but not K562 cells. 5' HS-3 may therefore contain information that contributes to the development-specific expression of the beta-like globin genes.     

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.     

Dosage-Dependent Gene Expression from Direct Repeat Locus in Rice Developed by Site-Specific Gene Integration

In the standard plant transformation practice, transgene copy number is often inversely correlated with transgene expression. As the integration locus generated by standard methods is mostly complex, consisting of both full-length and partial copies arranged in direct or inverted repeat configurations, it is difficult to parse the effect of copy number and locus structure. To clearly study the effect of transgene copy number on gene expression, it is important to control the locus structure and integrate full-length copies. In this study, the effect of transgene copy number on transgene expression in plant cells was determined using rice callus as a model. To generate full-length integrations, Cre-lox-mediated site-specific gene integration method was used. Transgenic rice lines consisting of one to three copies of β-glucuronidase or green fluorescent protein genes were developed. Site-specific integration lines were characterized and subjected to expression analysis. Lines containing two or three copies of either reporter genes displayed 2–4 times higher expression compared to the single-copy lines. Therefore, dosage-dependent transgene expression can be obtained by integrating full-length copies, and site-specific gene integration approach can serve as an efficient tool for generating precise multi-copy integrations.     

High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric beta-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment. In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay. The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct. Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold. In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA. GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than approximately 10 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increases expression without eliminating variation between transformants. We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.     

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。     

Transgene expression from CpG-reduced lentiviral gene delivery vectors in vitro

Current viral gene delivery vectors for gene therapy are inefficient due to short-lived transgene expression attributed to the cytosine-phosphate-guanine (CpG) motifs in the transgene. Here we assessed the effects of CpG motif reduction in lentiviral (LV) gene delivery context on the level and duration of reporter gene expression in Chinese Hamster Ovary (CHO) cells, Human Immortalized Myelogenous Leukemia (K562) cells and hematopoietic stem cells (HSCs). The cells were transduced with LV carrying Zero-CpG green fluorescent protein (ZGFP) reporter gene, LV/CMV/ZGFP. The GFP expression was compared to its non CpG-depleted GFP reporter gene LV (LV/CMV/GFP) counterpart. The LV/CMV/ZGFP exhibited prolonged transgene expression in CHO cells and HSCs up to 10 days and 14 days, in the respective cells. This effect was not seen in the transduced K562 cells, which may be due to the DNA hypomethylation status of the cancer cell line. Transgene copy number analysis verified that the GFP expression was not from pseudo-transduction and the transgene remained in the genome of the cells throughout the period of the study. The modest positive effects from the LV/CMV/ZGFP suggest that the reduction of CpG in the LV construct was not substantial to generate higher and more prolonged transgene expression.     

A method for determining zygosity of transgenic zebrafish by TaqMan real-time PCR

When producing a genetically modified organism, intended genes are often integrated into a target genome by random insertions. Subsequently, it is often desirable to know the gene copy number of the transgenic organism and the zygosity of its offspring. Because of the random insertions, the estimation can be made only by quantitative measurement of the genes. Even though TaqMan real-time PCR has been used in gene expression analysis, it is routinely used to quantify differences larger than twofold or more than one PCR cycle. In this study, we employed TaqMan quantitative PCR to determine zygosity of transgenic fluorescent zebrafish in which a homozygote and a hemizygote differ by only twofold. We measured relative quantities of the transgene by taking the threshold cycle (Ct) of both the transgene and an internal control zebrafish genomic DNA. Using scatterplots and statistical inference, we demonstrated that homozygotes and hemizygotes could be differentiated unambiguously when multiple measurements were taken. We discuss the relationship between the repetitive measurements and TaqMan precision with a statistical model. The result illustrates that the method can be extended to some areas that require even higher precision such as determining the polyploidy of an organism.