Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes

Previously, we established an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese hamster ovary (CHO) cell line. With a gradual increase in methotrexate (MTX) concentration, gene-amplified cell pools had high and stable specific growth and production rates. Moreover, the phenotype of gene-amplified cells seemed to be affected by the location of the amplified gene in chromosomal DNA. We suspected that various kinds of gene-amplified cells might appear during the long-term selection to construct gene-amplified cell pools. To clarify the behavior of gene-amplified cell pools during a stepwise increase of MTX concentration, we isolated gene-amplified clones derived from gene-amplified cell pools. We compared the characteristics of isolated clones, such as the productivity of recombinant protein, stability of amplified genes, and the location of amplified genes. As a result, telomere-type clones, in which the amplified gene was located near the telomeric region, were found to be more stable and productive than other types of clones. Telomere-type clones had over 100 copies of amplified genes in the chromosomal DNA. In contrast, a large number of other types of clones had less than 10 copies of amplified genes. During long-term cultivation in the absence of MTX, in other types of clones, amplified genes rapidly decreased in the chromosomal DNA.     

Flow cytometry: an improved method for the selection of highly productive gene-amplified CHO cells using flow cytometry.

In previous work, we clarified the relationship between the productivity and stability of gene-amplified cells and the location of the amplified gene. The location of the amplified gene enabled us to classify resistant cells into two types. One type of resistant cell group, in which the amplified genes were observed near the telomeric region, was named the "telomere type." The other type of cell group, in which the amplified genes were observed in other chromosomal regions, was named the "other type." The phenotypes of these two types of cells are very different. In this experiment, using a fluorescein isothiocyanate-labeled methotrexate (F-MTX) reagent with flow cytometry, we were easily able to distinguish between highly productive cells and the other types of cells. The level of fluorescence differed according to the difference in resistance to MTX. Based on this new finding, highly productive gene-amplified cells could be isolated from heterogeneous gene-amplified cell pools more easily than by the method of limiting-dilution assay. The limiting-dilution method requires several months to obtain highly productive gene-amplified cells, while our flow-cytometry-based method of selection requires only a few weeks.     

Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences.

A modular dihydrofolate reductase gene has been introduced into Chinese hamster ovary cells lacking dihydrofolate reductase. Clones capable of growth in the absence of added nucleosides contain one to five copies of the plasmid DNA integrated into the host genome. Upon stepwise selection to increasing methotrexate concentrations, cells are obtained which have amplified the transforming DNA over several hundredfold. A detailed analysis of the chromosomes in three clones indicated the appearance of cytologically distinct chromosomal regions containing the amplified plasmid DNA which differ in surrounding sequence composition, structure, and location. Two of the clones examined have extensive, homogeneously staining regions. The DNA in these homogeneously staining regions replicates in the early part of the S phase. The amplified plasmid DNA is found associated at or near the ends of chromosomes or on dicentric chromosomes. We propose that integration of DNA may disrupt telomeric structures and facilitate the formation of dicentric chromosomes, which may then undergo bridge breakage-fusion cycles. These phenomena are discussed in relation to DNA transfer experiments and modes of gene amplification and chromosome rearrangement.     

Limitations to the amplification and stability of human tissue-type plasminogen activator expression by Chinese hamster ovary cells

Chinese hamster ovary cell production of recombinant tissue-type plasminogen activator (t-PA) was increased by amplification of cotransfected dihydrofolate reductase cDNA using stepwise adaptation to increasing methotrexate (MTX) concentrations. The highest producing clones were isolated at 5 microM MTX and yielded 26,000 U/10(6) cells/day t-PA (43 microgram/10(6) cells/day). Above 25 microM MTX, cell specific t-PA production rates became increasingly variable and the cDNA copynumbers decreased. No apparent correlation between the cell specific t-PA production rate and the growth rate was observed upon subcloning of the amplified cells. When MTX selection was removed, the t-PA production rate decreased up to tenfold within 40 days; this was accompanied by an up to 60% drop in cDNA copynumber. Subclones isolated after 108 days of culture in the absence of MTX were, on average, sixfold more stable than their parental cells. In culture without MTX, the maximum stable t-PA production rate obtained (over 250 days) was 7000 +/- 750 U/10(6) cells/day (approximately 12 microgram/10(6) cells/day), approximately threefold lower than the maximum unstable levels of production reached under selective pressure. Taken together, these results define a wide range of the highest t-PA expression rates obtained under MTX selection, for which stable expression without selection has not been reported.     

Murine erythroleukemia cell variants: isolation of cells that have amplified the dihydrofolate reductase gene and retained the ability to be induced to differentiate

A series of murine erythroleukemia cell (MELC) variants was generated by selection for the ability to grow in increasing concentrations of the folate antagonist methotrexate (MTX). Growth of the parental MELC strain DS-19 was completely inhibited by 0.1 microM MTX. We isolated cells able to grow in 5, 40, 200, 400, and 800 microM MTX. Growth rates and yields were essentially the same in the presence or absence of the selective dose of MTX for all variants. MTX resistance was not the result of a transport defect. Dihydrofolate reductase (DHFR) from our variants and DS-19 was inhibited to the same extent by MTX. Variants had increased dihydrofolate reductase activities. The specific activity of DHFR was proportional to the selective concentration of MTX employed to isolate a given variant. DNA dot blotting established that the cloned variant (MR400-3) had a 160-fold increase in DHFR gene copy number relative to the parental strain (DS-19). Hybridization studies performed in situ established the presence of amplified DHFR genes on the chromosomes of the MTX-resistant but not the MTX-sensitive (parental) cells. Quantitation of DHFR mRNA by cytoplasmic dot blotting established that the amplified DHFR gene expression was proportional to gene copy number. Thus, MTX resistance was due to amplification of the DHFR gene. The variants retained the ability to be induced to differentiate in response to dimethyl sulfoxide and hexamethylenebis(acetamide) as evaluated by the criteria of globin mRNA accumulation, hemoglobin accumulation, cell volume decreases, and terminal cell division.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ubiquitous Chromatin Opening Elements (UCOEs) effect on transgene position and expression stability in CHO cells following methotrexate (MTX) amplification

The requirement for complex therapeutic proteins has resulted in mammalian cells, especially CHO cells, being the dominant host for recombinant protein manufacturing. In creating recombinant CHO cell lines, the expression vectors integrate into various parts of the genome leading to variable levels of expression and stability of protein production. This makes mammalian cell line development a long and laborious process. Therefore, with the intention to accelerate process development of recombinant protein production in CHO systems, UCOEs are utilized to diminish instability of production by maintaining an open chromatin surrounding in combination with MTX amplification. Chromosome painting and FISH analysis were performed to provide detailed molecular evaluation on the location of amplified genes and its relationship to the productivity and stability of the amplified cell lines. In summary, cell lines generated with vectors containing UCOEs retained stable GFP expression with MTX present (but instability was observed in the absence of MTX). UCOE cell lines displayed a higher frequency of integration into >1 chromosome than non‐UCOE group. Cell populations were more homogenous in terms of transgene location at the end of Long‐term culture (LTC). Overall our findings suggest variation in eGFP fluorescence may be attributed to changes in transgene integration profile over LTC.     

Cytogenetic analysis of chimeric antibody-producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure.

Previously, the highest producing (HP) recombinant CHO subclones isolated at various methotrexate (MTX) levels showed different antibody production stability during long-term culture, although they were clonally derived from CS13 transformant. In this study, genetic basis for their difference in antibody production stability was investigated using southern blot hybridization and fluorescence in situ hybridization (FISH) techniques. Southern analysis of HP subclones revealed that light-chain (LC) and heavy-chain (HC) cDNAs were located closely within 23 kb on an amplification unit, and the configuration of LC and HC cDNAs within this amplification unit was not disrupted during long-term culture in the absence of MTX. However, when LC and HC genes were localized on the metaphase chromosomes of HP subclones using FISH, the amplified sequences were present as an extended array on diverse marker chromosomes. HP subclones selected at higher MTX level had more kinds of marker chromosomes. CS13*-002 isolated at 0.02 microM MTX had only one marker chromosome (m002), whereas CS13*-1.0 isolated at 1 microM MTX had five different ones (m10A, m10B, m10C, m10D, and m10E). Each marker chromosome showed different fate during long-term culture of HP subclones in the absence of MTX, resulting in different degrees of stability among the HP subclones. The m10A and m10B remained unchanged, whereas the others disappeared or evolved to variants with shortened amplified arrays. The cells containing stable marker chromosomes constituted dominant subpopulations in CS13*-1.0, and thereby CS13*-1.0 became most stable in regard to antibody production during long-term culture. Furthermore, our dual-color FISH showed that the telomeric ends of amplified arrays on the stable marker chromosomes were always surrounded by (TTAGGG)(n) sequences, indicating that (TTAGGG)(n) sequences are closely related to the stability and evolution of amplified sequences. Taken together, our data show that the assessment of genotypic stability of amplified CHO cells is a prerequisite for understanding their production stability during long-term culture in the absence of selection pressure.     

Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells

Summary CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - EPSPS 5-enolpyruvylshikimate-3-phosphate synthase - I₅₀ 50% inhibitory concentration - Kb Kilobase (pairs) - PEP phosphoenolpyruvate - PMSF phenylmethylsulfonyl fluoride - PVPP polyvinylpolypyrrolidone - S-3-P shikimate-3-phosphate     

Coregulation of dihydrofolate reductase and thymidylate synthase in overproducer cell lines of wild carrot

Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities are associated with a 285,000 molecular weight enzyme complex in carrot (Daucus carota L.). Selection for methotrexate (MTX) resistance by stepwise increase of the concentration of MTX results in a high frequency adaptation to MTX with little or no significant increase in DHFR activity. However, when as a second step following MTX selection a specific inhibitor of TS, 5-fluoro-2-deoxyuridine was used, DHFR overproducer lines were obtained. The overproduction phenotype of the lines was almost completely lost after 8 weeks of growth in the absence of selection pressure. Although DHFR and TS are independent gene products, their activities increase in proportion (~20-fold) in the overproducer lines. This strongly suggests that DHFR and TS are not only functionally and physically linked in the same enzyme complex, but also are coregulated. These cell lines resemble the MTX-induced DHFR overproducer amplified cell lines of mammalian origin in their mode of selection, high frequency of appearance, elevated enzyme activity, and increased specific mRNA levels.     

Cytogenetic analysis in Polypterus ornatipinnis (Actinopterygii, Cladistia, Polypteridae) and 5S rDNA

Polypteridae is a family of archaic freshwater African fish that constitute an interesting subject for the study of the karyological evolution in vertebrates, on account of their primitive morphological characters and peculiar relationships with lower Osteichthyans. In this paper, a cytogenetic analysis on twenty specimens of both sexes of Polypterus ornatipinnis the ornate "bichir", coming from the Congo River basin, was performed by using both classical and molecular techniques. The karyotypic formula (2n=36; FN=72) was composed of 26 M+10 SM. The Alu I banding, performed to characterize heterochromatin in this species, was mainly centromeric. Both the chromosome location of the ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA(3) staining and FISH. CMA(3) marked all centromerical regions and showed the presence of two GC rich regions on the p arm of the chromosome pair n°1 and on the q arm of the pair n°14. Staining with Ag-NOR marked the only telomeric region of the chromosome n°1 p arm. After PCR, the 5S rDNA in this species was cloned, sequenced and analyzed. In the 665bp 5S rDNA sequence of P.ornatipinnis, a conserved 120bp gene region for the 5S rDNA was identified, followed by a non-transcribed variable spacer (NTS) which included simple repeats, microsatellites and a fragment of a non-LTR retrotransposon R-TEX. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair n°14, previously marked by CMA(3). FISH with 18S rDNA marked the telomeric region of the p arm of the pair n°1, previously marked both by Ag-NOR and CMA(3). The (GATA)(7) repeats marked the telomeric regions of all chromosome pairs, with the exclusion of the n°1, n°3 and n°14; hybridization with telomeric probes (TTAGGG)(n) showed signals at the end of all chromosomes. Karyotype evolution in Polypterus genus was finally discussed, including the new data obtained.     

Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase gene

Summary We have selected a tobacco cell line, SU-27D5, that is highly resistant to sulfonylurea and imidazolinone herbicides. This line was developed by selection first on a lethal concentration of cinosulfuron and then on increasing concentrations of primisulfuron, both sulfonylurea herbicides. SU-27D5 was tested against five sulfonylureas and one imidazolinone herbicide and was shown, in every case, to be two to three orders of magnitude more resistant than wild-type cells. The acetohydroxyacid synthase (AHAS) of SU-27D5 was 50- to 780-fold less sensitive than that of wild-type cells to herbicide inhibition. The specific activity of AHAS in the SU-27D5 cell lysate was 6 to 7 times greater than that in wild-type cells. Using Southern analysis, we showed that cell line SU-27D5 had amplified its SuRB AHAS gene about 20-fold while maintaining a normal diploid complement of the SuRA AHAS gene. Genomic clones of both AHAS genes were isolated and used to transform wild-type tobacco protoplasts. SuRB clones gave rise to herbicide-resistant transformants, whereas SuRA clones did not. DNA sequencing showed that all SuRB clones contained a point mutation at nucleotide 588 that converted amino acid 196 of AHAS from proline to serine. In contrast, no mutations were found in the SuRA clones. The stability of SuRB gene amplification was variable in the absence of selection. In one experiment, the withdrawal of selection reduced the copy number of the amplified SuRB gene to the normal level within 30 days. In another experiment, amplification remained stable after extended cultivation on herbicide-free medium. This is the first report of amplification of a mutant herbicide target gene that resulted in broad and strong herbicide resistance.     

Karyotypic characterization and genomic organization of the 5S rDNA in <Emphasis Type="Italic">Polypterus senegalus</Emphasis> (Osteichthyes,Polypteridae)

Polypteridae (Cladistia) is a family of archaic fishes, confined to African freshwaters. On account of their primitiveness in anatomical and morphological characters and mosaic relationships among lower Osteichthyans fishes, they constitute an important subject for the study of evolution in vertebrates. Very little is known about the karyological structure of these species. In this article, a cytogenetic analysis on twenty specimens of Polypterus senegalus (Cuvier, 1829) was performed using both classical and molecular techniques. Karyotype (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG) _n , (GATA)₇ repeats and ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA₃ staining and FISH. Staining with Ag-NOR showed the presence of two GC rich NORs on the p arm of the chromosome pair no. 1. CMA₃ marked all centromerical and some (no. 1 and no. 14) telomeric regions. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair no. 14. FISH with 18S rDNA marked the telomeric region of the p arm of the chromosome pair no. 1, previously marked by Ag-NOR. (GATA)₇ repeats marked the subtelomeric regions of all chromosome pairs, with the exclusion of the no. 1, 3 and 14. Hybridization with telomeric probes (TTAGGG) _n showed bright signals at the end of all chromosomes. After cloning, the 5SrDNA alignment revealed an organization of sequences made up of two different classes of tandem arrays (5S type I and 5S type II) of different lengths.     

Localization of amplified CAD genes on rearranged chromosomes of Chinese hamster cells

Chinese hamster cell lines carrying an amplified CAD region were selected from V79,B7 cells by their resistance to N-phosphonacetyl-L-aspartate (PALA). In one of the selected cell lines, SP PALA _inf1 ^supR L, an acrocentric chromosome with abnormally elongated q arms was identified as a marker for the PALA-resistant phenotype. The marker chromosome carried a homogeneously staining region close to a telomeric nucleolar organizer region. In the same region, localization of amplified CAD sequences was demonstrated by in situ hybridization. The marker chromosome was found to undergo extensive rearrangements. In particular, dicentric chromosomes, occurring with an unusually high incidence, were found to originate from end-fusion of two homologous marker chromosomes.Abbreviations ATCase Aspartate Transcarbamylase - CAD Carbamyl-phosphate synthetase-Aspartate transcarbamylase-Dihydroorotase - MTX Methotrexate - NOR Nucleolar Organizer Region - PALA N-phosphonacetyl-L-Aspartate     

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

Summary Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.     

Clonal variability within dihydrofolate reductase-mediated gene amplified Chinese hamster ovary cells: stability in the absence of selective pressure

Recombinant Chinese hamster ovary (rCHO) cells expressing a high level of chimeric antibody were obtained by cotransfection of heavy- and light-chain cDNA expression vectors into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level up to 1.0 microM. To determine the clonal variability within the amplified cell population in regard to antibody production stability, 20 subclones were randomly isolated from the amplified cell population at 1.0 microM MTX (CS13-1.0 cells). Clonal analysis showed that CS13-1.0 cells were heterogeneous with regard to specific growth rate (mu) and specific antibody productivity (qAb), although they were derived from a single clone. The mu and qAb of 20 subclones were in the range of 0.51 to 0.72 day-1 and 10.9 to 19.1 microgram/10(6) cells/day, respectively. During 8 weeks of cultivation in the absence of selective pressure, the mu of most subclones did not change significantly. On the other hand, their qAb decreased significantly. Furthermore, the relative decrease in qAb varied among subclones, ranging from 30% to 80%. Southern and Northern blot analyses showed that this decreased qAb resulted mainly from the loss of amplified immunoglobulin (Ig) gene copies and their respective cytoplasmic mRNAs. For the sake of screening convenience, an attempted was made to correlate the initial properties of subclones (such as mu, qAb, and Ig gene copies) with their antibody production stability during long-term culture. Among these initial properties examined, only qAb of subclones could help to predict their stability to some extent. The subclones with high qAb were relatively stable with regard to antibody production during long-term culture in the absence of selective pressure (P < 0. 005, ANOVA). Taken together, the clonal heterogeneity in an amplified CHO cell population necessitates clonal analysis for screening stable clones with high qAb.     

Effect of amplification ofdhfr andlac Z genes on growth and β-galactosidase expression in suspension cultures of recombinant CHO cells

Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human β-interferon (β-IFN) or thelac Z gene which codes for β-galactosidase (β-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10⁻⁷, and 10⁻⁶ M methotrexate (MTX), and the β-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplifieddhfr gene content and foreign β-gal gene expression in the cell populations. A biochemical assay for β-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10⁻⁷ M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10⁻⁶ M MTX was 20% lower than that of recombinant CHO cells at 10⁻⁷ M MTX. There was no effect of 10⁻⁵ M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect ofdhfr and foreign gene amplification and increased β-galactosidase expression.     

Telomere-homologous sequences occur near the centromeres of many tomato chromosomes

Several bacteriophage lambda clones containinginterstitialtelomererepeats (ITR) were isolated from a library of tomato genomic DNA by plaque hybridization with the clonedArabidopsis thaliana telomere repeat. Restriction fragments lacking highly repetitive DNA were identified and used as probes to map 14 of the 20 lambda clones. All of these markers mapped near the centromere on eight of the twelve tomato chromosomes. The exact centromere location of chromosomes 7 and 9 has recently been determined, and all ITR clones that localize to these two chromosomes map to the marker clusters known to contain the centromere. High-resolution mapping of one of these markers showed cosegregation of the telomere repeat with the marker cluster closest to the centromere in over 9000 meiotic products. We propose that the map location of interstitial telomere clones may reflect specific sequence interchanges between telomeric and centromeric regions and may provide an expedient means of localizing centromere positions.     

Molecular cytogenetic characterization of telomere-specific repetitive DNA sequences in Rana rugosa

We performed a molecular cloning of the glutamic oxaloacetic transaminase (GOT1) gene from R. rugosa, and determined its chromosomal location. This gene was reportedly localized near the sex-determining region of the ZW sex chromosomes in the frog Buergeria buergeri; however, the GOT1 gene was mapped to the distal end of chromosome 9 in R. rugosa using a GOT1 cDNA FISH probe. This was also the case when a 46.3?kb genomic clone containing exon 8 and 9 and the 3'-neighboring region of the GOT1 gene, designated clone B, was used as probe. However, weak signals were also detected at the telomeric ends of other autosomes and the Z sex chromosome, and near the centromeric region of the W sex chromosome. To intensify the signals, we used eight internal fragments in clone B and applied them to chromosome mapping. Consequently, only two fragments containing repeated sequence blocks produced hybridization signals; those signals were observed on autosomes and ZW sex chromosomes. The 3'-neighboring region contained two types of repeated sequence elements: a 41?bp element, designated 41-REL, localized to telomeric ends of autosomes and a 31?bp element, designated 31-REL, localized to telomeric ends of all autosomes and the ZW sex chromosomes, and also near the centromere on the W long arm. The results collectively suggest that the two repeated sequence elements were independently amplified around the chromosomal telomeres in R. rugosa, indicating that they will be useful cytogenetic markers for studying karyotypic evolution-especially the W chromosome differentiation-in this species.