Efficient modification of the APRT gene by FLP/FRT site-specific targeting

The FLP/FRT site-specific recombination system was established and characterized at the APRT gene in CHO cells. Targeting frequencies with FLP-stimulation were about 1 to 5×10^–5, which were 6–22-fold above gene targeting frequencies in the absence of FLP. Fifty two APRT⁺ cell lines were analyzed by Southern blotting: 56% were FLP-targeted integrants; 33% were APRT target convertants; 11% gave undefined patterns. In separate experiments we first enriched for integrants by screening for two additional markers carried on the targeting vector; 18 of 19 (95%) of the resulting cell lines were integrants. Intrachromosomal site-specific recombination was tested by reexposing integrants to FLP. Intrachromosomal popouts were stimulated over 200-fold, while homologous recombination in an adjacent interval was unchanged. The utility of this system was demonstrated by one-step FLP targeting to generate chromosomal substrates for homologous recombination, and by a two-step, FLP-and-run procedure to construct a chromosomal substrate for illegitimate recombination.     

Targeted mutagenesis by homologous recombination in D. melanogaster

We used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo. These molecules undergo homologous recombination with the corresponding chromosomal locus to generate targeted alterations of the host genome. The results address several questions about the general utility of this technique. We show that genes not near telomeres can be efficiently targeted; that no knowledge of the mutant phenotype is needed for targeting; and that insertional mutations and allelic substitutions can be easily produced.     

Increased copy number at 17q22-q24 by CGH in breast cancer is due to high-level amplification of two separate regions

Studies by comparative genomic hybridization (CGH) have defined a chromosomal site at 17q22-q24 that is often overrepresented in breast cancer, neuroblastoma, and several other tumor types. Due to the limited resolution and dynamic range of CGH, it remains unclear whether this gain reflects high-level amplification of small subregion(s) or low-level gain of most of the distal 17q. We used 32 physically mapped 17q probes to construct more accurate copy number profiles for 14 breast cancer cell lines by interphase fluorescence in situ hybridization (FISH). Six cell lines (43%) showed an increased copy number of the 17q22-q24 region by CGH, and seven (50%) by FISH. FISH copy number profiles had a substantially higher dynamic range than did CGH profiles. FISH revealed two independent, highly amplified regions (A and B) at 17q23, separated by about 5 Mb of non-amplified DNA. These regions were distinctly telomeric from the ERBB2 gene locus. However, region A was often co-amplified with ERBB2, whereas B was amplified in cell lines that showed no ERBB2 amplification. We conclude that distal 17q gains recently discovered in breast cancer by CGH are due to high-level amplifications of two different regions at 17q23. This chromosomal region has previously been reported to undergo allelic loss and therefore was thought to harbor a tumor suppressor gene. The present FISH data provide support for the presence, and a starting point for the positional isolation, of 17q23 genes whose upregulation by amplification may play a role in the progression of breast cancer and many other tumor types. Genes Chromosomes Cancer 20:372–376, 1997. © 1997 Wiley-Liss, Inc.     

Homologous recombination in the tandem calmodulin genes of Trypanosoma brucei yields multiple products: compensation for deleterious deletions by gene amplification

Homologous recombination between a calmodulin-neomycin-resistance fusion gene and the Trypanosoma brucei chromosome takes place not only in the large 5'- and 3'-flanking segments of the calmodulin locus but also in any of the four tandem genomic calmodulin genes. This results in a recombined locus consisting of the chimeric neor gene and four, three, two, one, or zero functional calmodulin genes. Cells bearing this latter event have half of their normal number of intact calmodulin genes and an accompanying phenotype of slow growth. Over months of propagation, these lines acquire additional calmodulin genes, frequently by amplifying a calmodulin gene at the untargeted locus, and concomitantly revert to normal growth rate. This response could be related to the property of the trypanosome of maintaining most housekeeping genes in tandem chromosomal arrays. Recombination appears to be initiated by a crossover event between the linearized end of the transfecting plasmid and a homologous region in the host genome; the second crossover generally occurs internally and in that region requires no more than 87 bp of homology.     

Fluorescence in situ hybridization analysis shows the frequent occurrence of 14q32.3 rearrangements with involvement of immunoglobulin switch regions in myeloma cell lines

In many B-cell malignancies, 14q32.3 chromosomal rearrangements involving the immunoglobulin heavy chain (IgH) locus have been shown to be pathognomonic for the disease. Although in myeloma heterogeneous and complex karyotypes are found, 14q32.3 translocations are prominent. However, owing to the telomeric position of the IgH locus, 14q32.3 translocations may be easily missed. We established fluorescence in situ hybridization (FISH) assays on chromosomes and DNA fibers to determine both the occurrence of 14q32.3 rearrangements in myeloma cell lines and the precise localization of the breakpoints in the IgH locus. Our results show that 14q32.3 chromosomal rearrangements are present in almost every myeloma cell line analyzed (17 of 19, 89%). Breakpoint analysis of the lines harboring one or more 14q32.3 rearrangements with the use of fiber-FISH revealed the involvement of switch regions in the IgH locus in 11 of 17 cell lines. Remarkably, pseudogamma genes without switch regions were involved in 3 of 17 cell lines, all derived from IgA myelomas. Three of 17 cell lines contained breakpoints outside a switch or immunoglobulin heavy chain constant region. The almost ubiquitous presence of 14q32.3 rearrangements suggests an obligatory role in the development of myeloma. The high incidence of breakpoints involving switch regions indicates an oncogenic event in a late stage of B-cell differentiation.     

Heterogeneous pattern of chromosomal breakpoints involving the MYC locus in multiple myeloma

Chromosomal rearrangements of the MYC locus, which often involve the IG loci, are recurrent events in multiple myeloma (MM) and plasma cell leukemia (PCL). We used dual-color fluorescence in situ hybridization (FISH) to characterize the breakpoint locations of chromosomal translocations/rearrangements involving the MYC locus at 8q24 found in a panel of 14 MM cell lines and 70 primary tumors (66 MM and 4 PCL). MYC locus alterations were observed in 21 cases: MYC/IG (mainly IGH@) fusions in 11 cell lines and three patients (2 MM and 1 PCL), and extra signals and/or abnormal MYC localizations in seven patients (5 MM and 2 PCL). Fourteen of these cases were investigated by FISH analyses by use of a panel of BAC clones covering about 6 Mb encompassing the MYC locus. The breakpoints were localized in a region 100-250 kb centromeric to MYC in four cases, a region 500-800 kb telomeric to the gene in four cases, and regions > or = 2 Mb centromeric or telomeric to MYC in five cases. Two different breakpoints were detected in the KMS-18 cell line, whereas the insertion of a MYC allele was found in a complex t(16;22) chromosomal translocation in the RPMI8226 cell line. Our data document a relatively high dispersion of 8q24 breakpoints in MM.     

用定点整合系统表达抗基孔肯亚病毒人-鼠嵌合抗体的研究

目的:构建含有FRT序列的CHO/dhfr-工程细胞株,并在此细胞株中用定点整合系统表达抗基孔肯亚病毒人鼠嵌合抗体。方法:首先克隆FRT序列与HBsAg的融合基因FRT-HBsAg,然后将FRT-HBsAg亚克隆到pCI neo的MCS中,构建表达载体pCI FRT-HBsAg。以此载体转染CHO/dhfr-细胞,用1g/L的G418筛选抗性克隆。检测抗性克隆HBsAg的表达,筛选表达量高的克隆作为宿主细胞株,命名为CHO/dhfr-FRT+。将表达质粒pA FRTHFLF和pOG44以1∶9的质量比混合,取2μg转染CHO/dhfr-FRT+。转染48h后,换选择培养基(撤掉H、T)用96孔板进行克隆化,2wk后对长成的单克隆进行检测。筛选正确整合到CHO/dhfr-FRT+细胞中FRT位点的克隆,并不断提高MTX的浓度,进一步筛选表达量更高的克隆。在MTX的浓度为2×10-7mol/L时,获得稳定表达细胞株,其最后的表达量为5mg/L。对此细胞株扩大培养后,收集上清并纯化,以纯化的嵌合抗体进行SDS-PAGE及Westernblot和抗原结合活性的检测。结果:构建了高转录活性位点整合FRT序列的CHO/dhfr-细胞株,并在此细胞株中表达抗基孔肯亚病毒人鼠嵌合抗体,表达量为5mg/L。结论:构建了含有FRT序列的CHO/dhfr-工程细胞株,并在此细胞株中用定点整合系统表达抗基孔肯亚病毒人鼠嵌合抗体,为用此系统表达抗体分子和其他蛋白分子奠定了基础     

Establishment and characterization of chromosomal aberrations in human cholangiocarcinoma cell lines by cross-species color banding

Cholangiocarcinoma (CC), a malignant neoplasm of the biliary epithelium, is usually fatal because of difficulty in early diagnosis and lack of availability of effective therapy. Furthermore, little is known about the genetics and biology of CC. Only a few reports concerning cytogenetic studies of CC have been published, and few cell lines have been established. We recently established four CC cell lines, designated as SCK, JCK, Cho-CK, and Choi-CK, and report the first application of cross-species color banding (RxFISH) and multiple chromosome painting for the characterization of the chromosomal rearrangements of these CC cell lines. Each cell line had unique modal karyotypic characteristics and showed a variable number of numerical and structural clonal cytogenetic aberrations. Chromosomes 3, 6, 7, 8, 12, 14, 17, and 18 were commonly involved in structural abnormalities. Homogeneously staining regions were determined in SCK and JCK, and double minute chromosomes were found in Cho-CK. The chromosomal aberrations of the four CC cell lines were effectively analyzed by RxFISH and FISH with multiple chromosome painting probes. The nonrandom rearrangements suggest candidate regions for isolation of genes related to CC.     

Molecular cytogenetic analysis of a human breast metastasis model: identification of phenotype-specific chromosomal rearrangements

We have previously characterized an experimental system in which the role of candidate metastasis-related genes can be screened and tested. Monoclonal cell lines M4A4 and NM2C5 originated from the MDA-MB-435 breast tumor cell line but have opposite metastatic capabilities in vivo. To investigate gross genetic changes present in this model, we performed a detailed molecular cytogenetic evaluation of the parental cell line, the M4A4 and NM2C5 cell lines, and related clones of metastatic phenotype. Using a combination of spectral karyotyping (SKY), G-banding, and fluorescence in situ hybridization (FISH), we were able to resolve the identity of all common marker chromosomes present in MDA-MB-435 cells, and to define several chromosomal changes, which were specific to each monoclonal cell line. Twenty identical structural and numerical chromosomal aberrations, including trisomies of chromosomes 2 and 5 as well as t(1;7), t(1;10), t(8;11), t(8;15), and t(20;21), were present in all cell lines. The majority of translocations were relatively simple non-reciprocal rearrangements, most frequently involving chromosomes 19, 1, 6, and 20. Chromosomal gains of 1, 7q, 8q, and 20q are common alterations in breast cancer. The metastatic M4A4 cell line contained numerous unique chromosomal aberrations, of which an abnormal banding region on chromosome 22, abr(22), was the best clone-specific identifier. Conversely, the t(12;15)(q22;q26.1) was found exclusively in the non-metastatic NM2C5 cell line. The integration of these karyotypic data with other cytogenetic and genomic databases will enhance our ability to identify genes that play critical roles in cancer development and progression.     

Characterization of a chromosomally complex lung cancer cell line using multiwell fluorescence in situ hybridization

The chromosomal characterization of a non-small cell lung cancer cell line (NCIH358) is described. This characterization was achieved using a simple, cheap and technically straightforward multiwell fluorescence in situ hybridization (FISH) method. The many and complex chromosome rearrangements identified by this method could not be defined using conventional G-banded chromosome analysis, and have not been previously described. For the detailed characterization of complex cell lines, multiwell FISH has many advantages over more technically demanding and expensive FISH techniques, and opens up the possibility of screening for consistent rearrangements, leading to the identification of unique fusion genes.     

稳定表达狂犬病病毒CTN-1V株糖蛋白细胞系的建立

目的构建能稳定表达狂犬病病毒糖蛋白的CHO细胞系。方法用RT—PCR法扩增和分离CTN-1V株狂犬病毒糖蛋白基因,测序后克隆入pCDNA5．0FRT载体,构建重组质粒pCDNA5．0FRT—G,之后与POG44质粒共转染CHO细胞,采用潮霉素B抗性筛选,挑选阳性克隆,间接免疫荧光和WesternBlot进行稳定表达细胞系的鉴定。结果经酶切和测序鉴定,获得重组表达质粒pCDNA5．0FRT—G,共转染CHO细胞后,获得肉眼可见阳性细胞克隆,刮取阳性克隆进行传代扩大培养并定义为第2代,经过10代后免疫荧光和WesternBlot结果依然阳性。结论成功建立稳定表达狂犬病病毒糖蛋白的CHO细胞系,为深入研究糖蛋白的功能奠定基础。     

稳定表达UT-A2的FRT细胞系的建立与鉴定

目的：建立稳定表达尿素通道蛋白A2（UT—A2）的FRT细胞系，为寻找UT—A2抑制剂提供细胞模型。方法：通过真核表达质粒-脂质体介导的方法，将UT—A2 cDNA与真核表达载体pUB6／V5连接后的重组质粒转染入FRT细胞，经稳定筛选建立稳定表达UT—A2的FRT细胞系。功能检测实验将细胞分为对照组和稳定转染组，用2mol／L尿素负荷试验检测稳定表达UT—A2的FRT细胞膜的尿素通透性。结果：经BSD筛选21d后得到稳定表达UT—A2的细胞株；Western blotting证实UT—A2蛋白稳定表达；免疫荧光分析结果提示UT—A2的质膜定位。2mol／L尿素试验证实了该细胞系具有明显尿素通透性。结论：在非肾脏上皮细胞获得了稳定质膜定位表达UT—A2的FRT细胞株，该细胞株可用于UT—A2抑制剂的筛选。     

Different coexisting genotypes in the breast cancer cell line MDA-MB-468

Intratumor genetic heterogeneity, a well-known characteristic of numerous cancers, often confounds a precise diagnosis and leads to therapy resistance. This study deals with such chromosomal variability, which may be due to an inherent genetic instability affecting heterogeneity and clonal effects. Subpopulations of the breast cancer cell line MDA-MB-468 were isolated according to epidermal growth factor receptor (EGFR) expression by FACS. Whole genome profiling (CGH; mapping arrays) and determination of egfr gene amplification (fluorescence in situ hybridisation, FISH; qPCR) were done directly after sorting or after several passages of cell culture. Subpopulations differed in the amplification of the egfr-locus 7p11-14 showing egfr gene amplification rates of up to 60-fold in high-level expressing populations and less than 2-fold in low-level expressing populations. However, after several passages the original low-level cells showed a new amplification of the egfr gene, which was as heterogeneous as the original amplification detected in MDA-MB-468. Additional, spontaneously expressed fragile sites could be shown in FISH analyses which may affect cell culture heterogeneity. Understanding the precise chromosomal process would clarify mechanisms in vivo and improve both diagnosis and therapy of corresponding cancers.     

Establishment of a new human malignant fibrous histiocytoma cell line,FU-MFH-1: cytogenetic characterization by comparative genomic hybridization and fluorescence in situ hybridization

Although a number of malignant fibrous histiocytoma (MFH) cell lines have been reported, their characterization at a molecular cytogenetic level has not been fully established. In this study, we established a new human cell line, designated as FU-MFH-1, from a storiform-pleomorphic MFH arising in the retroperitoneum of a 61-year-old woman, and applied comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) with chromosome painting probes for the characterization of chromosome alterations. FU-MFH-1 cells were spindle, round, or polygonal in shape with oval nuclei, and were maintained continuously in vitro for over 50 passages for more than 12 months. G-banding analysis was performed and FU-MFH-1 revealed a complex karyotype with an abnormal chromosome 19 containing a homogeneously staining region (hsr). CGH analysis showed a high-level amplification of 12q13-->q21. The high-level amplification detected by CGH was refined by FISH. These results showed that the hsr was composed of amplified DNA sequences from 12q. Our study emphasizes the usefulness of CGH as a powerful tool for chromosomal localization of amplified sequences. The FU-MFH-1 cell line should be useful for biologic and molecular pathogenetic investigations of human MFH.     

Molecular cytogenetic characterization of rhabdomyosarcoma cell lines

Alveolar rhabdomyosarcomas (ARMS) are soft-tissue tumors that are genetically characterized by the presence of reciprocal translocations that generate the fusion gene PAX3-FOXO1A or PAX7-FOXO1A. For the study of the biologic consequences of such rearrangements, several cell lines have been generated. However, established cell lines accumulate chromosome and genetic aberrations that make it difficult to draw significant conclusions. We have applied a set of techniques that includes spectral karyotyping, fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and microarray CGH, to the most commonly used cell lines carrying the two fusion genes that are present in ARMS. We have identified the bacterial artificial chromosomes that cover the breakpoints at genes PAX3, PAX7, and FOXO1A, which can be used as FISH probes for the translocations. The RH30 cell line, positive for the PAX3-FOXO1A fusion gene, was found to be highly complex: wide range of chromosome number, more than 50 chromosome rearrangements, amplification of the hybrid gene, 24 DNA changes detected by conventional CGH, and 21 gene copy changes detected by microarray CGH (including several high-level amplifications). RMZ-RC2 cell line, positive for the PAX7-FOXO1A, was in the near-tetraploid range with only nonclonal structural rearrangements, amplification of the hybrid gene, 24 DNA changes by CGH, and 8 gene copy changes, confirming the previously reported high-level amplification of MYCN.     

Chromosomal location and structure of amplicons in two human cell lines with coamplification of c-myc and Ki-ras oncogenes

Gene amplification is a major mechanism through which oncogenes and genes responsible for drug resistance are overexpressed in neoplastic cells, and several models for structure of amplified units (amplicons) are postulated. In order to identify consistent changes associated with oncogene amplification, we analyzed chromosomal location and physical distance of amplicons of two independent human cell lines that have coamplified c-myc and Ki-ras oncogenes. In one cell line, KHC287, amplified c-myc genes were localized in two chromosomes and Ki-ras in three chromosomes. One marker chromosome was almost entirely encompassed by both amplified genes. In the other cell line, Lu-65, both of the amplified genes shared the same locus, on chromosome 12q+. The two genes, however, are more than 1500 kb apart in both cell lines. The above findings indicate that two different amplified genes became associated on one chromosome in two independent cell lines. This suggests that a common mechanism is associated with chromosomal rearrangements affecting different amplified genes.     

Liver metastatic ability of human melanoma cell line is associated with losses of chromosomes 4, 9p21-pter and 10p

Genetic changes underlying the aggressive progression of human cutaneous melanoma are not completely understood. In order to characterise genetic alterations associated with the metastatic behaviour of this neoplasm we used comparative genomic hybridisation (CGH) in combination with fluorescence in situ hybridisation (FISH) on an experimental metastatic model of three related human melanoma cell lines. Tumour lines were selected based on their various metastatic capacity to liver in immunosuppressed mice. The parental cell line (A2058) was a human amelanotic melanoma cell line, adaptation of this line to in vivo growth as xenograft the HT168 tumour and its cell line was established. After intrasplenic transplantation of HT168 cells into immunosuppressed mice, a highly metastatic variant (HT168-M1) was selected. Several chromosomal aberrations common to all three lines indicating common clonal origin, as well as additional non-shared chromosomal changes were found. The original cell line (A2058) exhibited the highest number of genetic changes. Chromosomal alterations present only in the highly metastatic line (HT168-M1) involved losses on chromosome 4, 9p21.3-pter and 10p. Chromosome copy number patterns and the nature of chromosome 4 loss were further investigated by FISH using different centromeric probes and a chromosome 4 painting probe. According to our CGH and FISH results we assume that alterations present only in the aggressive metastatic subline are associated with the increased – metastatic potential. Our observations further support the hypothesis, based on some recently published data, that certain (so far unidentified) suppressor genes having an important role in tumour progression are located on these chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.