Establishment of transformed swine fibroblast cell lines using SV40 large T antigen

Summary Swine testicle cell lines were established by transformation of primary swine testicle (PST) cells with an SV40 plasmid (pSV3-neo), which contains genes conferring resistance to neomycin and expressing SV40 large T antigen. Plasmid DNA was transfected into PST cells using a lipofection system. Two related plasmids, pSV2-neo and pSV5-neo, failed to induce transformed cells. Cells transformed with pSV3-neo formed single colonies that were resistant to the antibiotic, G418, and expressed large T antigen. Upon two cycles of cloning by endpoint dilution method, three transformed clones, designated transformed swine testicle (tST)-3, tST-14 and tST-18, were selected and characterized in regards to cell replication and susceptibility to swine viruses. The resultant clones were compared with a counterpart non-transformed ST cell line (ATCC-ST). The three tST cell lines showed longer or the same doubling times and higher saturation densities compared to ATCC-ST cells. These cells were free from a range of adventitious agents and supported the replication of porcine parvovirus (PPV), pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV), comparable to ATCC-ST cells. All three cell lines have been maintained in continuous cultures for over 60 passages with no changes in growth characteristics. These findings indicate that lipofection with pSV3-neo is an efficient means for the introduction of exogenous DNA into porcine cells and for establishment of transformed immortalized cell lines.     

Regulation of G418 selection efficiency by cell-cell interaction in transfection

We attempted to establish the optimum conditions for the calcium phosphate (CaPO₄) precipitation protocol by counting G418 resistant (G418^r) colonies after transfection of pSV2-neo DNA into BALB 3T3 cells. The amount and molecular size of carrier DNA, number of plating cells, treatment period of DNA-CaPO₄ precipitates and expression time of G418 selection were found to be important factors in the induction of G418^r colonies. Six G418^r clones were derived from BALB 3T3, NIH 3T3 and FRSK cells, and cocultured with G418 sensitive (G418^s) parent cells in G418 medium. The colony formation capacity of all G418^r cell clones decreased with the increasing number of plated G418^s cells. Cell-cell contact appeared to be necessary to reduce the colony formation of G418^r cells, and contact-dependent G418^r cell killing was probably not related to gap junction formation. Contact-mediated cell killing is a likely explanation for the observation that induction of G418^r colonies is often reduced under conditions of high-density plating, long treatment of DNA-CaPO₄ precipitates, and long expression time of G418 selection. These results suggest that in some instances transfection efficiency using pSV2-neo DNA should be carefully evaluated because culture conditions can mask the induction of G418^r colonies.     

Somatic cell hybrid selection with a transfectable dominant marker

A recombinant plasmid vector, pSV2-neo, coding for resistance to neomycin and the related antibiotic G-418, was transfected into the mouse myeloma line X63-Ag8.653 by a modification of the protoplast fusion technique. The time interval required to obtain 10⁶ G-418 resistant cells was 20 days and the efficiency was 10^–4–10^–5, which represents a significant advantage over classical methods of selecting mutant cells bearing a dominant selection marker. To investigate the efficiency of this marker in somatic cell hybrid selection, these cells were fused to the human myeloma line U-266 and the hybrids were selected either in HAT + G-418 or HAT + ouabain. The pSV2-neo vector was as efficient as ouabain as a dominant marker with respect to the number of viable hybrid colonies selected and their levels of immunoglobulin secretion. The reciprocal experiment was also performed: HAT-sensitive, mutant U-266 cells were transfected with pSV2-neo, clones selected in G-418 and fused with X63-Ag8.653 cells, and hybrids selected in ouabain plus G-418, yielding HAT-sensitive hybrid heteromyelomas that were effective fusion partners with human B lymphocytes.     

Cosmids and transcribed sequences from chromosome 11q23

Summary To obtain cosmid markers and transcribed sequences from a specific chromosome region, a series of radiation-reduced hybrids (RHs) containing various regions of human chromosome 11 was prepared from microcell hybrid A9 (neo11) cells containing a normal human chromosome 11 tagged with pSV2neo at 11p11.2. Among 15 radiation hybrid clones isolated, RH(11)-9 which contains a q23 fragment in addition to theneo integration site, was used for the construction of a cosmid library. Cosmid clones having human DNA sequences were screened, and localized by Southern hybridization with the radiation hybrid panel. Fifty-nine cosmids were assigned to 11q23 and 6 cosmids to 11p11.2. Exon amplification proceeded with 23 of the 59 cosmids and 16 putative exons were cloned. Three of them were identical to those constituting a known gene which locates on q23 (ATDC), and the others were unknown. Thus, the RHs containing various subchromosomal fragments of chromosome 11 were useful for constructing region-specific DNA markers. The RH-(11)-9 cells and putative exons also facilitate the positional cloning of genes in the 11q23 region.     

Suppression of endometrial carcinoma cell tumorigenicity by human chromosome 18

Presumptive tumor suppressor genes may be localized to specific chromosomes by the procedure of microcell fusion, whereby individual chromosomes derived from normal human cells are introduced into tumor cells. Allelic loss on chromosome I 8 is commonly seen in endometrial carcinoma, and the DCC gene on chromosome arm 18q is a potential human tumor suppressor gene. In this study, we investigated the hypothesis that a gene on chromosome 18, possibly DCC, is capable of suppressing the tumorigenicity of endometrial carcinoma cells. Microcells from the mouse A9 cell clone containing one human chromosome I8 tagged with the pSV2-neo plasmid were fused with the highly tumorigenic endometrial carcinoma cell lines HHUA and Ishikawa, and G418-resistant microcell hybrids containing an extra copy of chromosome I8 were isolated. Clones isolated from the HHUA cell line were completely suppressed for tumorigenicity in nude mice, and clones from the lshikawa line were suppressed or inhibited for tumorigenicity. In contrast, growth rates in vitro were not significantly affected in clones from either parental cell line. DCC expression was elevated in most of the suppressed hybrids. These results indicate that a gene on human chromosome I 8 is capable of suppressing the tumorigenicity of endometrial carcinoma cells, and that DCC is a candidate for this endometrial carcinoma tumor suppressor gene. © 1995 Wiley-Liss, Inc.     

The use of human repetitive DNA to target selectable markers into only the human chromosome of a human-hamster hybrid cell line (AL)

We used the plasmid BLUR-8 that contains an 800-base pair (bp) sequence of human repetitiveAlu DNA in a cotransfection protocol to target the plasmids pSV2neo or EBO-pcD-leu-2 (hygro) into a single site of the sole human chromosome, number 11, of a Chinese hamster-human hybrid cell line (A_L). The neo and hygro plasmids confer resistance to antibiotics G418 and hygromycin, respectively. Of the 33 cotransfected clones with single-site insertions, 1/13 without BLUR-8 and 6/20 with BLUR-8 were only in human chromosome 11. A frequency of insertion of 1/13 is not different than expected by chance (=0.3512). On the other hand, the probability that 6/20 insertions, as seen with BLUR-8, occurred by chance is low (=0.0003). We suggest that the human DNA sequences contained in BLUR-8 targeted insertions into only the human chromosome.     

Restoration of radiation resistance in ataxia telangiectasia cells by the introduction of normal human chromosome 11

In order to identify the human chromosome which carries a mutated gene in cells from a patient with the hereditary disorder ataxia telangiectasia belonging to complementation group D (AT-D), we performed chromosome transfer experiments via microcell fusion. A single, pSV2neo-tagged chromosome, either 11 or 12, derived from normal human fibroblasts was introduced into AT-D cells by microcell fusion, and clones which were resistant to the antibiotic G418 were isolated. All 3 hybrid clones containing an additional copy number of chromosome 11 showed a restoration of the resistance of wild-type cells to killing by X-irradiation, whereas all 3 hybrid clones containing an additional copy number of chromosome 12 remained hyper-radiosensitive, like the parental AT cells. The results indicate that a defective gene of AT-D cells is also located on chromosome 11, since a genetic linkage analysis has previously suggested that a defective gene of its complementation group A is located on this chromosome.     

Modification of Bacterial Artificial Chromosome Clones Using Cre Recombinase: Introduction of Selectable Markers for Expression in Eukaryotic Cells

Bacterial artificial chromosome clones (BACs) are widely used at present in human genome physical mapping projects. To extend the utility of these clones for functional genomic studies, we have devised a method to modify BACs using Cre recombinase to introduce a gene cassette into the loxP sequence, which is present in the vector portion of the BAC clone. Cre-mediated integration is site specific and thus maintains the integrity of the genomic insert sequences, while eliminating the steps that are involved in restriction digest-based DNA cloning strategies. The success of this method depends on the use of a DNA construct, RETRObac, which contains the reporter marker green fluorescent protein (GFP) and the selectable marker neomycin phosphotransferase (neo), but does not contain a bacterial origin of replication. BAC clones have been modified successfully using this method and the genomic insert shows no signs of deletions or rearrangements. Transfection efficiencies of the modified BACs into human or murine cell lines ranged from 1% to 6%. After culture in media containing G418 for 3 weeks, ~0.1% of cells previously sorted for GFP expression acquired stable antibiotic resistance. Introduction of a human BAC clone that contains genomic p53 sequences into murine NIH3T3 cells led to expression of human p53 mRNA as determined by RT–PCR, demonstrating that sequences contained on the BAC are expressed. We believe that GFP–neo modified BAC clones will be a valuable resource in efforts to study biological effects of known genes as well as in efforts to clone and analyze new genes and regulatory regions.     

Increased efficiency of transfection of murine hybridoma cells with DNA by electropermeabilization

Dispase-treated murine hybridoma cells (SP2/0-Ag14) were transfected with the G418 resistance gene bearing plasmid pSV2-neo by electropermeabilization with a high degree of efficiency. The cells were subjected to intermittent multiple high-voltage short duration (5 microsecond) DC pulses at intervals of 1 min in a weakly conducting medium followed by selection in G418-containing medium. The transfection medium, temperature, pulse duration, and voltage were empirically determined by preliminary electropermeabilization experiments. Increasing the number of pulses resulted in a higher percentage of transfected cells, but a decrease in the number of viable cells, with the optimal transfectant yield resulting when five pulses of 10 kV/cm were administered. This method allows the rapid and efficient injection of DNA into mammalian cells, and permits the rapid production of stable, drug resistant hybridoma cell lines for use in subsequent fusion experiments.     

Identification of human gene complementing ts AlS9 mouse L-cell defect in DNA replication following DNA-mediated gene transfer

The temperature-sensitive (ts) mouse L-cell, ts AlS9, is defective in a gene required for nuclear DNA replication early in the S phase of the cell cycle. Human DNA sequences were introduced into ts AlS9 cells together with the plasmid pSV2neo, which can confer resistance to the drug geneticin. Cotransformants, expressing both the plasmid-derived neomycin gene and the transferred human AlS9 gene, were selected for growth in the presence of the drug at the nonpermissive temperature (npt). The resulting transformants retained a common set of human-specific Alu repetitive DNA sequences. These are likely to be accommodated within, or in proximity to, the transferred human AlS9 gene. The results obtained provide the basis for cloning human genes required for DNA replication.     

De novo retrotransposition of unbiased sequences in a human breast cancer cell clone.

It has been demonstrated recently that certain repetitive sequences and even expressed single-copy genes are capable of retrotransposition, but little is known about the endogenous or exogenous modifiers of this process in human cells. Retrotransposition may contribute to gene inactivation and genetic instability in cancer development. We have used the human cell line MCF-7 to generate a method for investigating de novo retrotransposition in breast cancer cells. The strategy employs a reporter construct transfected into MCF-7 cells that encodes neomycin phosphotransferase gene (neoR) sequences interrupted by an intron derived from the gamma-globin gene and sandwiched between two promoters in opposite orientation; the phosphotransferase is not produced in transfected cells expressing the plasmid until transposition via a spliced antisense neoR RNA intermediate has occurred, conferring a functional gene product and thereby resistance to G418. A stable transfectant line that showed presence of reporter plasmid DNA and expression of reporter antisense neoR was obtained and used to demonstrate spontaneous retrotransposition of neoR sequences: tester cells were subjected to selection in G418 medium, and neomycin-resistant clones were isolated at a frequency of 10(-7). A simple PCR-based prescreening of colonies fixed and stained in Petri dishes can be used to verify intronless neoR DNA. Expanded populations of G418-resistant colonies were determined to be derived from reporter sequences that had transposed via an RNA intermediate by Southern blot genotyping. This experimental assay may be used for exploring endogenous and environmental factors that influence host cell-mediated retrotransposition of unbiased cellular sequences in breast tumor cells. Genes Chromosomes Cancer 26:84-91, 1999.     

Comparative FISH mapping of the ancestral fusion point of human chromosome 2

It is known that human chromosome 2 originated from the fusion of two ancestral primate chromosomes. This has been confirmed by chromosome banding and fluorescence in-situ hybridization (FISH) with human chromosome-2-specific DNA libraries. In this study, the order of 38 cosmid clones derived from the human chromosome region 2q12-q14 was exactly determined by high-resolution FISH in human chromosome 2 and its homologous chromosomes in chimpanzees (Pan trogrodydes, 2n=48) and cynomolgus monkeys (Macaca fascicularis, 2n=42). This region includes the telomere-to-telomere fusion point of two ancestral ape-type chromosomes. As a result of comparative mapping, human chromosome region 2q12-q14 was found to correspond to the short arms of chimpanzee chromosomes 12 and 13 and cynomolgus monkey chromosomes 9 and 15. It is noted that no difference was detected in the relative order of the cosmid clones between human and chimpanzee chromosomes. This suggests that two ancestral ape-type chromosomes fused tandemly at telomeres to form human chromosome 2, and the genomic organization of this region is thought to be considerably conserved. In the cynomolgus monkey, however, the order of clones in each homologue was inverted. In addition to cosmid mapping, two chromosome-2-specific yeast artificial chromosome (YAC) clones containing the fusion point were identified by FISH. This revised version was published online in July 2006 with corrections to the Cover Date.     

High-efficiency stable gene transfection using chloroquine-treated Chinese hamster ovary cells

We describe a highly efficient stable gene transfection procedure for Chinese hamster ovary (CHO) cells using a modification of the calcium phosphate-DNA coprecipitation method. We have found that treatment of CHO cells with chloroquine increases the efficiency of gene transfer by up to 20-fold (from approx. 0.01% to approx. 0.2%) when transfection is done using the pSV2-neo plasmid. The optimized transfection procedure requires that CHO cells to be incubated with calcium phosphate-DNA coprecipitate and chloroquine (100 µM) for a total of 16 h. By using high-molecular-weight human genomic DNA as a DNA source for transfection, we show that this procedure is equally efficient for stably transferring a much larger gene, such as the 49-kb human hypoxanthine phosphoribosyltransferase gene.     

Physical map of a YAC contig containing the region of the human gene (HYRC) complementing hyper-radiosensitivity of the scid mouse mutation

Summary We previously mapped the putative humanHYRC (the hyper-radiosensitivity of the scid mutation, complementing gene) to human chromosome 8q11.1 by fluorescencein situ hybridization (FISH) using Alu-based PCR products from a mouse-human scid radiation cell hybrid (RD15/5) as probes. From a cosmid library constructed from RD15/5, 57 cosmid clones containing human DNA inserts were isolated, 18 of which were mapped to 8q11. Based on the sequences of plasmid subclones of the 18 cosmids, five novel sequence-tagged-sites (STSs) were made. By a screening of the CEPH-YAC library with these STSs, five yeast artificial chromosome, (YAC) clones were isolated. All these YAC clones were confirmed not to be chimeric by FISH, but two of them showed deleted human insert DNAs. Using the other 3 non-deleted YACs, we constructed a physical map covering theHYRC region. We confirmed that the recently isolated gene (the DNA-PK_cs gene) which is a strong candidate forHYRC is located within the present contig and spans less than 200 kb. This map will be useful for the analysis of the genomic structure of the DNA-PK_cs gene and for isolation of other complementing genes in theHYRC region.     

Reactivation of psoralen-reacted plasmid DNA in Fanconi anemia,xeroderma pigmentosum,and normal human fibroblast cells

We have used a host cell reactivation system to study the effect of 8-methoxypsoralen (8-MOP) reaction on CAT (chloramphenicol acetyltransferase) and NEO (aminoglycoside phosphotransferase) expression in normal human cells, as well as two cell lines with possible DNA repair-processing defects. Plasmid DNA was treated with psoralen plus near-ultraviolet (NUV) irradiation. The reacted plasmids, pSV2cat and pSV2neo, were transfected into Fanconi anemia (FA), xeroderma pigmentosum (XP), and normal human fibroblast cells for transient or stable assay. The cells were assayed for CAT activity at various times after transfection or selected for G418 resistance. The extent of adduct formation required to inhibit expression was much less (difference of D₃₇ greater than 2.5) in FA or XP cells compared to normal. We conclude that in FA and XP cells, the reactivation of CAT was much less than in normal cells. The possibility of differential DNA uptake and/or degradation in transient assay was ruled out by analysis of plasmid DNA recovered from transfected cells. The data of the two independent assays indicate that FA and XP cells are deficient in cross-linked DNA repair.     

EB病毒膜蛋白在CHO细胞中的表达

目的研制ＥＢ（Ｅｐｓｔｅｉｎ－Ｂａｒ）病毒基因工程疫苗。方法构建了含有Ｅｐｓｔｅｉｎ－Ｂａｒ病毒（ＥＢＶ）膜蛋白（ＭＡ）基因的重组表达质粒ｐＣＭＶ／ＭＡ，转染ＣＨＯ细胞，研究表达产物的生物学性状，及培养液中不同血清含量、冻存、Ｇ４１８对ＣＨＯ细胞分泌ＭＡ的影响。结果获得两个稳定表达ＭＡ的细胞株。免疫印迹检测表达产物的分子量约为３４０ｋＤ和２２０ｋＤ，间接免疫荧光和免疫斑点确定表达产物能与抗ＭＡ的单克隆抗体特异性结合，用薄层扫描和Ｌｏｗｒｙ法计算ＭＡ的表达量为每天１９μｇ／ｍｌ。经纯化的ＭＡ免疫小鼠，在血清中检测到抗ＭＡ的特异性抗体。结论为开发有效的表达系统用于ＥＢ病毒基因工程疫苗的生产提供有利的实验基础。     

Correction of a nucleotide-excision-repair mutation by human chromosome 19 in hamster-human hybrid cells

A UV-sensitive mutant line of CHO cells, UV20, was shown to be phenotypically corrected to resistance by fusion with human lymphocytes or fibroblasts. Only human chromosome 19 correlated with the DNA repair phenotype of resistant hybrid clones and their resistant or sensitive subclones. This study demonstrates the mapping of a human repair gene by direct selection of complementing hybrids in the presence of a DNA-damaging agent (mitomycin C).     

Somatic cell hybridization of Roberts syndrome and normal human fibroblasts transfected with plasmids carrying dominant selection markers

Roberts syndrome (RS) is a rare human recessive disorder involving, in the chromosomes of some patients, a characteristic puffing or splitting apart of the constitutive heterochromatin (the RS effect). We carried out somatic cell hybridizations between an RS cell strain (R22) with the heterochromatin abnormality and a hypoxanthine phosphoribosyltransferase-deficient cell strain (GM1662) with normal chromosome structure to determine if the presence of the normal genome would correct the RS effect in the hybrid cells. In order to provide the fibroblast strains with dominant selection markers for the hybridizations, GM1662 was transfected with the plasmid pSV3neo which conferred resistance to the antibiotic G418, and R22 was transfected with the plasmid pSV3gpt which provided resistance to mycophenolic acid. Two somatic cell hybridizations were carried out: (1) R22×GM1662 pSV3neo and (2) R22 pSVgpt×GM1662 pSV3neo. The RS effect was found to be absent in 95% and 92%, respectively, of the 200 hybrid cells examined in each experiment. This indicated that the GM1662 genome was able to correct the RS effect. The presence of the RS effect in a few of the hybrid cells was attributed to the unstable karyotype resulting from pSV3 transfection which presumably caused the loss of the normal allele(s) of the RS gene in these hybrid cells.     

High-frequency transfection of CHO cells using polybrene

High-frequency transfection of CHO cells has been achieved for several plasmids, a cosmid library, and genomic DNA using Polybrene and dimethyl sulfoxide. All plasmid transfectants examined were stable and exhibited plasmid sequences in genomic DNA. The method is simple, reproducible, and succeeded with several independent CHO clones in the presence or the absence of carrier DNA, even at very low concentrations of plasmid DNA.