Non-expressing Clones Research Articles

An IRES-Mediated Tricistronic Vector for Efficient Generation of Stable, High-Level Monoclonal Antibody Producing CHO DG44 Cell Lines.

The generation of stable, high-level monoclonal antibody (mAb) producing cell lines remains a major challenge in biopharmaceutical industry. The commonly used plasmid vectors for mAb expression, which express light chain (LC), heavy chain (HC), and selection marker genes on separate vectors or via multiple promoters on a single vector, are not able to accurately control the ratio of LC over HC expression and tend to result in non-expressing clones. To overcome these issues, we have developed a tricistronic vector using two internal ribosome entry sites (IRES) to express the LC, HC, and dihydrofolate reductase (DHFR) selection marker genes in one transcript. In this tricistronic vector, the three genes are under the control of a hapten-modified human cytomegalovirus (hCMV) promoter containing a core CpG island element (IE) to enhance the production stability. The LC gene is arranged as the first cistron followed by a wild-type IRES to control the HC expression. Such design expresses excess LC polypeptides which enhance mAb expression level and reduce aggregate. A mutated IRES with attenuated strength is applied on DHFR to reduce its expression for enhancing the stringency of selection for high producers. This vector allows easy generation of stable, high mAb producing CHO DG44 pools and clones for antibody development and manufacturing.

NS1′ protein expression facilitates production of Japanese encephalitis virus in avian cells and embryonated chicken eggs

Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus of the family Flaviviridae, is a leading cause of meningo-encephalitis in Asian countries. The flavivirus non-structural protein 1 (NS1) plays a role in virus replication and in the elicitation of an immune response. The NS1' protein found among the members of the JEV subgroup is an extended form of NS1 and is generated by a -1 ribosomal frameshift. This protein is known to be involved in viral pathogenicity; however, its specific function is still unknown. Here, we describe an investigation of the molecular function of NS1' protein through the production of JEV NS1'-expressing and -non-expressing clones and their infection of avian and mammalian cells. Efficient NS1' protein expression was observed in avian cells and was found to facilitate JEV production in both avian cultured cells and embryonated chicken eggs. NS1' protein was observed to co-localize with NS5 protein and resulted in increased viral RNA levels in avian cells. These findings clearly indicate that NS1' enhances the production of JEV in avian cells and may facilitate the amplification/maintenance role of birds in the virus transmission cycle in nature.

Analysis of heterogeneity and instability of stable mAb-expressing CHO cells

Screening and isolation of high expression mammalian cell lines for production of recombinant proteins for the clinic is a resource-intensive and time-consuming procedure due to the substantial variation in expression levels of recombinant protein expression amongst transfected cells. Several investigators have reported instability in expression titers early in cell line development and in cell banks. However, in most cases the exact molecular mechanisms of instability remain unknown. In this study we used a fluorescence-activated cell sorting (FACS) based mAb staining method to enable the detection and selective gating of cells with vastly different recombinant expression levels present in transfected pools. Expression diversity and changes within transfected populations were detected and isolated in real time during cell line development. Molecular genetic analysis on the isolated clones revealed an unsuspected rearrangement of the heavy chain in the non-expressing clones. Implications of the genetic rearrangements as well as the use of the FACS method as a tool to improve cell line development to detect expression heterogeneity in pools and to investigate root cause for the molecular genetics of expression instability will be discussed.

IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines

A Tricistronic vector utilizing internal ribosome entry site (IRES) elements to express the light chain (LC), heavy chain (HC), and a neomycin phosphotransferase (NPT) selection marker from one transcript is designed for generation of mAb expressing CHO cell lines. As compared to the commonly used vectors, benefits of this design include: (1) minimized non-expressing clones, (2) enhanced stable mAb productivity without gene amplification, (3) control of LC and HC expression at defined ratios, and (4) consistent product quality. After optimization of the LC and HC arrangement and increasing selection stringency by weakening the NPT selection marker, this Tricistronic vector is able to generate stably transfected pools with specific productivity (qmAb) greater than 5pg/cell/day (pcd) and titers over 150mg/L. 5% of clones from these pools have qmAb greater than 20pcd and titers ranging from 300 to more than 500mg/L under non-optimized shake flask batch cultures using commercially available protein-free medium. The mAb produced by these clones have low aggregation and consistent glycosylation profiles. The entire process of transfection to high-expressing clones requires only 6 months. The IRES-mediated Tricistronic vector provides an attractive alternative to commonly used vectors for fast generation of mAb CHO cell lines with high productivity.

HCHL expression in hairy roots of Beta vulgaris yields a high accumulation of p-hydroxybenzoic acid (pHBA) glucose ester, and linkage of pHBA into cell walls

As part of a study to explore the potential for new or modified bio-product formation, Beta vulgaris (sugar beet) has been genetically modified to express in root-organ culture a bacterial gene of phenylpropanoid catabolism. The HCHL gene, encoding p-hydroxycinnamoyl-CoA hydratase/lyase, was introduced into B. vulgaris under the control of a CaMV 35S promoter, using Agrobacterium rhizogenes LBA 9402. Hairy root clones expressing the HCHL gene, together with non-expressing clones, were analysed and revealed that one expression-positive clone accumulated the glucose ester of p-hydroxybenzoic acid (pHBA) at about 14% on a dry weight basis. This is the best yield achieved in plant systems so far. Determination of cell-wall components liberated by alkaline hydrolysis confirmed that the ratio of pHBA to ferulic acid was considerably higher in the HCHL-expressing clones, whereas only ferulic acid was detected in a non-expressing clone. The change in cell-wall components also resulted in a decrease in tensile strength in the HCHL-expressing clones.

Epigenetic mechanisms are involved in the control of Giardia lamblia antigenic variation

Giardia lamblia, an intestinal dwelling protozoan parasite, undergoes surface antigenic variation where only one of an estimated 150 variant-specific surface proteins (VSPs) is expressed and present on the surface at any one time. Transcriptional switching between VSPs results in replacement of one VSP by another. The mechanisms that control antigenic variation are poorly understood and difficult to study because there are multiple copies of each VSP and strong similarity with other VSPs. In order to study transcriptional regulation of one specific vsp, a haemagglutinin (HA) epitope-tagged h7 was integrated into the G. lamblia GS genome. We show that HA-tagged H7 undergoes antigenic variation in the same manner as native H7, also present in the GS genome. Control of expression of both HA-tagged H7 and native H7 is independent of each other even though the genes and their surrounding 5' and 3' flanking sequences are virtually identical. Analysis of expressing and non-expressing clones revealed an absence of HA-tagged h7 gene rearrangements upon switching and acetylation of histone lysine residues within the 167 nucleotides 5' to the expressed HA-tagged h7 gene. Lack of vsp rearrangements and acetylation of expressed immediate upstream regions implicates involvement of epigenetic mechanisms in antigenic variation.

Methyl-CpG Binding Domain Protein 2 Represses Transcription from Hypermethylated π-Class Glutathione S-Transferase Gene Promoters in Hepatocellular Carcinoma Cells

During the pathogenesis of human hepatocellular carcinoma (HCC), the CpG island encompassing the pi-class glutathione S-transferase gene (GSTP1) becomes hypermethylated. Repression of transcription accompanying CpG island hypermethylation has been proposed to be mediated by methyl-CpG binding domain (MBD) proteins. We report here that inhibition of transcription from hypermethylated GSTP1 promoters in Hep3B HCC cells, which fail to express GSTP1 mRNA or GSTP1 polypeptides, appears to be mediated by MBD2. Treatment of Hep3B cells with 5-azadeoxycytidine (5-aza-dC), a methyltransferase inhibitor, activated GSTP1 expression, whereas treatment with trichostatin A, a histone deacetylase inhibitor, had little effect. To more precisely assess the contribution of the pattern of GSTP1 CpG island methylation on GSTP1 mRNA expression, Hep3B cells were treated for 72 h with 5-aza-dC and then subjected to limiting dilution cloning. Bisulfite sequencing was used to map the methylation patterns of the GSTP1 promoter region in GSTP1-expressing and -non-expressing clones. In the clone that expressed GSTP1 mRNA determined by Northern blot analysis and quantitative reverse transcriptase (RT)-PCR, widespread demethylation of at least one GSTP1 allele was evident. Chromatin immunoprecipitation experiments revealed the presence of MBD2, but not Sp1, at the GSTP1 promoter in Hep3B cells. In contrast, Sp1 was detected at the GSTP1 promoter in a GSTP1-expressing Hep3B 5-aza-dC subclone. To test whether MBD2 might be responsible for the inhibition of GSTP1 transcription from hypermethylated GSTP1 promoters, siRNAs were used to reduce MBD2 polypeptide levels in Hep3B cells. SssI-catalyzed methylation of GSTP1 promoter sequences resulted in diminished luciferase reporter activity after transfection into Hep3B cells. However, when hypermethylated GSTP1 promoter sequences were transfected into Hep3B cells that had been treated with siRNA-targeting MBD2 mRNA, no repression of luciferase reporter expression was evident. These findings implicate MBD2 in the repression of GSTP1 expression associated with GSTP1 CpG island hypermethylation in HCC cells.

Ectopic expression of cyclin D1 impairs the proliferation and enhances the apoptosis of a murine lymphoid cell line.

Cyclin D1, a key regulator of the cell cycle, acts as an oncogene when over-expressed in several types of cancer. In some B-chronic lymphoproliferative disorders, the over-expression of cyclin D1 protein is thought to confer a proliferative phenotype. We have generated BaF3 pro-B cell derivatives in which cyclin D1 can be induced rapidly and reversibly in a dose-dependent manner by the hormone muristerone A. When non-expressing clones displayed the same proliferative capacity as the parental cell line, in the sub-clones, a moderate induction of cyclin D1 lengthened the proliferation rate. The over-expression of cyclin D1 had the same effects on cell proliferation but also led ultimately to cell death by apoptosis. The induction of cyclin D1 in growth factor-deprived cells as well as in anticancer drug-treated cells also reinforced the magnitude of apoptosis. Thus, the expression of cyclin D1 in lymphoid cells does not confer a proliferative advantage but rather alters the response of cells towards apoptotic stimuli in a p53-independent manner.

The 3′-Untranslated Region of the α2C-Adrenergic Receptor mRNA Impedes Translation of the Receptor Message

We report that two subtypes of alpha2-adrenergic receptors (alpha2A/D- and alpha2C-AR) are ectopically expressed with dramatically different efficiencies and that this difference is due to a 288-nucleotide (nt) segment in the 3'-untranslated region (3'-UTR) of the alpha2C-AR mRNA that impairs translational processing. NIH-3T3 fibroblasts were transfected with receptor constructs (coding region plus 552 nt, alpha2C-AR; coding region plus 1140 nt, alpha2A/D-AR) and a vector conferring G418 resistance. Transcription was driven by the murine sarcoma virus promoter element, and the receptor gene segment was upstream of an SV40 polyadenylation cassette. Drug-resistant transfectants were evaluated for expression of receptor mRNA and protein. 90% of the NIH-3T3 alpha2C-AR transfectants expressed receptor mRNA, but only 14% of the clonal cell lines expressed receptor protein. In contrast, 90% of the NIH-3T3 alpha2A/D-AR transfectants expressed receptor protein (200-5000 fmol/mg). Similar results were obtained following transfection of DDT1MF-2 cells with the two receptor constructs. The role of the 3'-UTR of the alpha2C-AR in mRNA processing was determined by generating new constructs in which the 3'-UTR was progressively truncated from 552 to 470, 182, 143, or 74 nt 3' to the stop codon. Truncation of the 3'-UTR resulted in the expression of receptor protein in the G418-resistant transfectants (nt 74, 100%; nt 143, 80%; nt 182, 50%). The level of mRNA in the transfectants expressing the receptor protein was not greater than that in nonexpressing clones, and the differences in protein expression did not reflect altered mRNA stability in the truncated construct. The alpha2C-AR mRNA with the longer 3'-UTR underwent translational initiation as it was found in the polysome fraction, indicating that the lack of receptor protein was due to impaired translational elongation or termination. These data suggest that translational efficiency is a key mechanism for regulating alpha2C-AR expression and associated signaling events.

Engineering hyperexpression of bacteriophage Mu C protein by removal of secondary structure at the translation initiation region.

The structure at the translation initiation region (TIR) of mRNA has pronounced regulatory effects on gene expression. Our attempts to overexpress the C gene of bacteriophage Mu in a variety of expression vectors resulted in low yields of protein. Analysis of Mu C mRNA shows the potential to form a secondary structure involving a ribosome binding site and AUG codon. We have engineered the overproduction of the protein using a PCR-aided cloning approach to remove the sequences involved in the formation of this secondary structure. The overexpressing clone, under the control of T7 gene 10 promoter in a T7 expression system yielded > 30% of total cell protein. The difference in mRNA structure between expressing and non-expressing clones was confirmed by electrophoretic analysis of run-off transcripts. The overexpressed protein was purified in a single step by site-specific DNA affinity chromatography. The purified recombinant protein was active in band shift assays. DNA binding activity required Mg2+ and was weak in the presence of Mn2+. Cd2+ or Zn2+ could not support DNA binding. Under optimal conditions, the equilibrium binding constant (Kapp) was determined to be 2 x 10(12) M-1.

Cloning, expression, and cDNA sequence of surface antigen P22 from Toxoplasma gondii

Immunoblot, immunofluorescence, and complement-mediated cytolytic assays revealed that two new monoclonal antibodies raised against a membrane-enriched fraction of Toxoplasma gondii tachyzoites recognize protein P22 on the surface of the parasite. Using these monoclonal antibodies to screen a cDNA expression library in λgt11, several clones expressing recombinant fusion proteins were isolated. Subsequent screening of the library with a synthetic oligonucleotide derived from the 5′ end of one of these cDNAs permitted the isolation of additional nonexpressing clones containing the entire translated sequence. Blots of parasite RNA and DNA suggested that the corresponding gene occurs as a single copy in the tachyzoite genome. The amino acid sequence deduced from the composite cDNA indicates a primary translation product with a theoretical molecular weight of 18 959. As expected for surface protein P22, the putative polypeptide contains a predicted N-terminal signal sequence and a C-terminal hydrophobic region characteristic of proteins attached to the membrane by a glycophospholipid anchor. Recombinant fusion proteins produced by the expressing clones were recognized on immunoblots by IgG antibodies in the sera of humans with acute and chronic T. gondii infection. Antibodies selected by the fusion protein reacted predominantly with a 22-kDa antigen on immunoblots of parasite lysate.

5-Azacytidine-induced reactivation of the human X chromosome-linked PGK1 gene is associated with a large region of cytosine demethylation in the 5' CpG island.

Hamster-human cell hybrids containing an inactive human X chromosome were treated with 5-azacytidine and derived clones were examined for phosphoglycerate kinase activity and cytosine methylation in the human PGK1 (X chromosome-linked phosphoglycerate kinase) gene. Comparisons between expressing and nonexpressing clones indicated that demethylation of several methylation-sensitive restriction sites outside of the 5' CpG island were unnecessary for expression. High-resolution polyacrylamide gel analysis of 25 Hpa II, Hha I, and Tha I sites revealed that all clones expressing PGK1 were unmethylated in a large region of the CpG island that includes the transcription start site and 400 base pairs upstream. Many nonexpressing clones had discontinuous patterns of demethylation. Remethylation was often observed in subclones of nonexpressing hybrids. These data suggest that a specific zone of methylation-free DNA within the PGK1 promoter is required for transcription. In addition, the presence of neighboring methylcytosines appears to decrease the heritable stability of unmethylated CpGs in this region.

Two distinct forms of surface antigen gene rearrangement in Trypanosoma brucei

African trypanosomes express different surface antigens sequentially in their mammalian hosts1. Expression of different variable surface glycoprotein (VSG) antigens is associated with rearrangements of genomic DNA2–5. Several laboratories have reported that expression of some VSG genes is accompanied by the appearance of a duplicated copy of the genes at a new location in the genome3,4. However, in a series of sequentially related Trypanosoma brucei clones from a different stock we were unable to detect duplication of one VSG gene (ILTAT 1.2, B) in expressing clones. Two copies of this gene were found in expressing and non-expressing clones. Apparent insertions and deletions occurred near the 3′ ends of both gene copies, but could not be correlated with expression2,5,6. We report here the observation of both types of rearrangement for different VSG genes in the same series of trypanosome clones. Thus the different types of rearrangement are not a function of different trypanosome stocks, but of different VSG genes within a stock.