Characterisation of recombinant CHO cell lines by investigation of protein productivities and genetic parameters

We have generated a recombinant CHO cell line expressing the fusion protein EpoFc. After selection and screening, protein expression, gene and mRNA copy numbers were analysed in order to gain more information on the influence of genetic parameters on the productivity and stability of production cells. Results from semi-quantitative blot methods were compared to quantitative PCR (qPCR) analyses, whose advantage mainly lies in their higher sensitivity, and the cheaper and faster methodology. We developed stable and high producing clones with low gene copy numbers, in contrast to other cell lines where multiple steps of methotrexate amplification have lead to hundreds of copies of inserts with the risk of karyotypic instabilities and decreased growth rates that overcome the benefits of increased productivities. When comparing genetic parameters to productivity, a good correlation of mRNA levels with specific productivity was observed, whereas high gene copy numbers were not always accompanied by high protein expressions. Based on our data derived from a typical example of a cell line development process, genetic parameters are useful tools for the selection of scalable production clones. Nevertheless, a wider range of cell lines has to be investigated in order to implement genetic analyses into a screening process.     

Transgene copy number comparison in recombinant mammalian cell lines: critical reflection of quantitative real-time PCR evaluation

Nucleic acid quantification is a relevant issue for the characterization of mammalian recombinant cell lines and also for the registration of producer clones. Quantitative real-time PCR is a powerful tool to investigate nucleic acid levels but numerous different quantification strategies exist, which sometimes lead to misinterpretation of obtained qPCR data. In contrast to absolute quantification using amplicon- or plasmid standard curves, relative quantification strategies relate the gene of interest to an endogenous reference gene. The relative quantification methods also consider the amplification efficiency for the calculation of the gene copy number and thus more accurate results compared to absolute quantification methods are generated. In this study two recombinant Chinese hamster ovary cell lines were analysed for their transgene copy number using different relative quantification strategies. The individual calculation methods resulted in differences of relative gene copy numbers because efficiency calculations have strong impact on gene copy numbers. However, in context of comparing transgene copy numbers of two individual clones the influence of the calculation method is marginal. Therefore especially for the comparison of two cell lines with the identical transgene any of the relative qPCR methods was proven as powerful tool.     

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.     

Determination of HSV-1 UL5 and UL29 gene copy numbers in an HSV complementing Vero cell line

The genetic stability of transgenes is a critical characteristic used to assess constructed cell lines used for vaccine production. The evaluation of gene copy numbers by a qPCR method, is one of the most common approaches used to assess the consistency of transgenes in a constructed cell line. The cell line AV529-19 is a Vero-based cell line specifically engineered to express the HSV-1 UL5 and UL29 open reading frames. AV529-19 is used to support the replication of a defective HSV-2 viral candidate vaccine called HSV529. To assess the genetic stability of the UL5 and UL29 transgenes in AV529-19 cells, a digital PCR-based approach was developed. During characterization of the test method, the specificity, accuracy, and intermediate precision of the assay was investigated based on regulatory guidelines. The developed assay was used to monitor the stability of the transgenes in the manufactured AV529-19 cell lines by comparison of transgene copy numbers in the master cell bank (MCB) with their copy numbers in the extended cell bank (ECB). Results showed that the UL29 and UL5 transgenes are stable in that there are one and three copies of the UL29 and UL5 genes, respectively, per cell in both the AV529-19 MCB and ECB.     

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     

Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis

A comprehensive genomic analysis of single cells is instrumental for numerous applications in tumor genetics, clinical diagnostics and forensic analyses. Here, we provide a protocol for single-cell isolation and whole genome amplification, which includes the following stages: preparation of single-cell suspensions from blood or bone marrow samples and cancer cell lines; their characterization on the basis of morphology, interphase fluorescent in situ hybridization pattern and antibody staining; isolation of single cells by either laser microdissection or micromanipulation; and unbiased amplification of single-cell genomes by either linker-adaptor PCR or GenomePlex library technology. This protocol provides a suitable template to screen for chromosomal copy number changes by conventional comparative genomic hybridization (CGH) or array CGH. Expected results include the generation of several micrograms of DNA from single cells, which can be used for CGH or other analyses, such as sequencing. Using linker-adaptor PCR or GenomePlex library technology, the protocol takes 72 or 30 h, respectively.     

Detection of novel amplicons in prostate cancer by comprehensive genomic profiling of prostate cancer cell lines using oligonucleotide-based arrayCGH

BACKGROUND: The purpose of this study was to prove the feasibility of a longmer oligonucleotide microarray platform to profile gene copy number alterations in prostate cancer cell lines and to quickly indicate novel candidate genes, which may play a role in carcinogenesis. METHODS/RESULTS AND FINDINGS: Genome-wide screening for regions of genetic gains and losses on nine prostate cancer cell lines (PC3, DU145, LNCaP, CWR22, and derived sublines) was carried out using comparative genomic hybridization on a 35,000 feature oligonucleotide microarray (arrayCGH). Compared to conventional chromosomal CGH, more deletions and small regions of gains, particularly in pericentromeric regions and regions next to the telomeres, were detected. As validation of the high-resolution of arrayCGH we further analyzed a small amplicon of 1.7 MB at 9p13.3, which was found in CWR22 and CWR22-Rv1. Increased copy number was confirmed by fluorescence in situ hybridization using the BAC clone RP11-165H19 from the amplified region comprising the two genes interleukin 11 receptor alpha (IL11-RA) and dynactin 3 (DCTN3). Using quantitative real time PCR (qPCR) we could demonstrate that IL11-RA is the gene with the highest copy number gain in the cell lines compared to DCTN3 suggesting IL11-RA to be the amplification target. Screening of 20 primary prostate carcinomas by qPCR revealed an IL11-RA copy number gain in 75% of the tumors analyzed. Gain of DCTN3 was only found in two cases together with a gain of IL11-RA. CONCLUSIONS/SIGNIFICANCE: ArrayCGH using longmer oligonucleotide microarrays is feasible for high-resolution analysis of chomosomal imbalances. Characterization of a small gained region at 9p13.3 in prostate cancer cell lines and primary prostate cancer samples by fluorescence in situ hybridization and quantitative PCR has revealed interleukin 11 receptor alpha gene as a candidate target of amplification with an amplification frequency of 75% in prostate carcinomas. Frequent amplification of IL11-RA in prostate cancer is a potential mechanism of IL11-RA overexpression in this tumor type.     

Structural conservation of the transposon Tam3 family in Antirrhinum majus and estimation of the number of copies able to transpose

We have investigated the organization of the transposon Tam3 family in Antirrhinum majus. Genomic hybridization experiments and characterization of 40 independent Tam3 clones isolated from an A. majus plant revealed that the Tam3 family is quite conserved and the copy sizes are uniform. We did not find any copy with a deleted internal sequence, unlike what is usually observed in other transposons. This exceptionally conserved structure of the Tam3 family was confirmed by PCR and sequencing analyses. Sequencing analysis identified eight copies with sequences completely identical to that of the Tam3 transposase gene. These results suggested that a considerable number of autonomous Tam3 copies are present in the genome of A. majus. Among 24 copies which are surrounded by single copy regions of the genome, 14 copies are present as specific insertions in the line which we used, but absent in other lines. These copies are therefore predicted to be movable. If this ratio is the same for all Tam3 copies in a genome, then a maximum of 60% of the copies are estimated to be movable in the genome. The relatively high frequency of gene tagged by Tam3 might reflect the large number of movable copies in the genome.