Beta-lactamase reporter system for selecting high-producing yeast clones

In modern production of protein biopharmaceuticals, a good screening and selection method of high-producing clones can dramatically influence the whole production process and lead to lower production costs. We have created a rapid, simple, and inexpensive method for selecting high-producing clones in the yeast Pichia pastoris that is based on the beta-lactamase reporter system. By integrating the reporter gene and the gene of interest into the same genome locus, it was possible to use beta-lactamase activity as a measure of the expression level of the protein of interest. A novel expression vector with two independent expression cassettes was designed and tested using green fluorescent protein (GFP) as a model. The first cassette contained the GFP gene under the control of a strong, inducible AOX1 promoter, while the second cassette consisted of the beta-lactamase reporter gene under the control of a weak constitutive YPT1 promotor. High-producing GFP clones were selected directly on the plates based on the color change after hydrolysis of the beta-lactamase substrate added to the medium. beta-lactamase activity was found to positively correlate with GFP fluorescence. The reporter system described is widely applicable-it can be easily applied to other, also pharmaceutically relevant proteins and to other yeast expression systems, such as Saccharomyces cerevisiae and Hansenula polymorpha.     

Construction and Application of an Inducible System for Homogenous Expression Levels in Bulk Cell Lines

Stringently controlled conditional expressing systems are crucial for the functional characterization of genes. Currently, screening of multiple clones to identify the tightly controlled ones is necessary but time-consuming. Here, we describe a system fusing Tet (tetracycline)-inducible elements, BAC (bacterial artificial chromosome) and Gateway technology together to allow tight control of gene expression in BAC-transfected eukaryotic bulk cell cultures. Recombinase cloning into the shuttle vector and the BAC facilitates vector construction. An EGFP (enhanced green fluorescent protein) allows FACS (fluorescence activated cell sorting) and the BAC technology ensures tight control of gene expression that is independent of the integrating site. In the current first application, our gene of interest encodes a β-catenin-ERα fusion protein. Tested by luciferase assay and western blotting, in HTB56 lung cancer cells the final BAC E11-IGR-β-catenin-ERα vector demonstrated sensitive inducibility by Tet or Dox (doxycycline) in a dose-dependent manner with low background, and the EGFP was an effective selection marker by FACS in bulk culture HTB56 and myeloblastic 32D cells. This is a highly efficient tool for the rapid generation of stringently controlled Tet-inducible systems in cell lines.     

Functional inhibition of transitory proteins by intrabody-mediated retention in the endoplasmatic reticulum

Intrabodies are recombinantly expressed intracellular antibody fragments that can be used to specifically bind and inhibit the function of cellular proteins of interest. Intrabodies can be targeted to various cell compartments by attaching an appropriate localization peptide sequence to them. An efficient strategy with a high success rate is to anchor intrabodies in the endoplasmatic reticulum where they can inhibit transitory target proteins by binding and preventing them to reach their site of action. Intrabodies can be assembled from antibody gene fragments from various sources into dedicated expression vectors. Conventionally, antibody cDNA sequences are derived from selected hybridoma cell clones that express antibodies with the desired specificity. Alternatively, appropriate clones can be isolated by affinity selection from an antibody in vitro display library. Here an evaluation of endoplasmatic reticulum targeted intrabodies with respect to other knockdown approaches is given and the characteristics of various intrabody expression vectors are discussed. A step by step protocol is provided that was repeatedly used to construct intrabodies derived from diverse antibody isotypes producing hybridoma cell clones. The inactivation of the cell surface receptor neural cell adhesion molecule (NCAM) by a highly efficacious novel endoplasmatic reticulum-anchored intrabody is demonstrated.     

PC-１基因表达增强C4-2B前列腺癌细胞生存

建立稳定表达外源PC-１基因的人前列腺癌骨转移C4-2B细胞模型,初步探讨PC- １基因表达对前列腺癌发展的影响.通过脂质体介导的方法,将融合PC-１基因的真核表达载体pcDNA3.1PC-１稳定转染C4-2B细胞,Western 印迹和RT-PCR技术,分别从蛋白水平和RNA水平确定外源PC-１基因表达. MTT和软琼脂集落形成能力等一系列方法,研究PC-１基因的功能,RT-PCR和实时定量PCR检测前列腺癌发生发展相关基因表达的变化. 结果表明,PC-１基因的高表达能够诱导雄激素受体（AR）调控基因和一系列重要的信号通路成员基因PSA、PSMA、NKX31、Jagged1、EphA3、SGEF和 NOTCH3等表达发生变化. 实验结果初步证明,PC-１基因表达在晚期前列腺癌中,以及在雄激素非依赖的转变中可以发挥作用,PC-１基因表达可调控一些重要信号通路.对PC-１基因功能深入研究将有可能为发现新的前列腺癌的诊断治疗分子靶标提供线索.     

Gene expression changes in human lung cells exposed to arsenic, chromium, nickel or vanadium indicate the first steps in cancer

The complex process of carcinogenesis begins with transformation of a single cell to favor aberrant traits such as loss of contact inhibition and unregulated proliferation - features found in every cancer. Despite cancer's widespread prevalence, the early events that initiate cancer remain elusive, and without knowledge of these events cancer prevention is difficult. Here we show that exposure to As, Cr, Ni, or vanadium (V) promotes changes in gene expression that occur in conjunction with aberrant growth. We exposed immortalized human bronchial epithelial cells to one of four metals/metalloid for four to eight weeks and selected transformed clonal populations based upon anchorage independent growth of single cells in soft agar. We detected a metal-specific footprint of cancer-related gene expression that was consistent across multiple transformed clones. These gene expression changes persisted in the absence of the progenitor metal for numerous cell divisions. Our results show that even a brief exposure to a carcinogenic metal may cause many changes in gene expression in the exposed cells, and that from these many changes, the specific change(s) that each metal causes that initiate cancer likely arise.     

Creation of genome-wide protein expression libraries using random activation of gene expression

Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 x 10(6) individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, approximately 53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.     

Physical mapping of yeast artificial chromosomes containing sequences from the human beta-globin gene region

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human beta-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult beta-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire beta-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the beta-globin gene cluster.     

Conservation and alteration of HLA-B27-specific T cell epitopes on mouse cells. Implications for peptide-mediated alloreactivity.

Alloreactive CTL responses generate a great variety of clonal specificities. Such diversity may be related to recognition of multiple peptides constitutively bound to any given MHC alloantigen. Among human alloreactive CTL, only a fraction of the clones lyse mouse P815 cells expressing class I HLA proteins. In this study the fine specificity of HLA-B27 allorecognition on human or mouse cells by five human HLA-B27-specific CTL clones was comparatively analyzed. This was done to examine what degree of variation in epitope structure is compatible with recognition of HLA Ag on mouse cells. Nine site-specific HLA-B27 mutants were expressed on both human and mouse cells, after DNA-mediated gene transfer, to construct two analogous series of target cells. The reaction patterns of four of the five CTL clones with these cell panels were compatible with conservation of their corresponding epitopes upon expression of HLA-B27 on mouse cells. The reaction pattern of the fifth clone was different with either cell panel, indicating that its epitope was structurally altered on mouse cells. It also suggested a selectively increased expression of the determinant on these cells. The results suggest that most of the epitopes recognized by allospecific CTL clones reacting across species are either independent of any bound peptide or involve identical peptides from both cell types. However, some of these clones recognize alloantigen-bound peptides that are somewhat different in structure depending on the cell type, and may be expressed at the mouse cell surface in greater amounts. Such peptides could arise from related proteins in both species, and be polymorphic as a result of phylogenetic divergence.     

The microarray explorer tool for data mining of cDNA microarrays: application for the mammary gland

The Microarray Explorer (MAExplorer) is a versatile Java-based data mining bioinformatic tool for analyzing quantitative cDNA expression profiles across multiple microarray platforms and DNA labeling systems. It may be run as either a stand-alone application or as a Web browser applet over the Internet. With this program it is possible to (i) analyze the expression of individual genes, (ii) analyze the expression of gene families and clusters, (iii) compare expression patterns and (iv) directly access other genomic databases for clones of interest. Data may be downloaded as required from a Web server or in the case of the stand-alone version, reside on the user’s computer. Analyses are performed in real-time and may be viewed and directly manipulated in images, reports, scatter plots, histograms, expression profile plots and cluster analyses plots. A key feature is the clone data filter for constraining a working set of clones to those passing a variety of user-specified logical and statistical tests. Reports may be generated with hypertext Web access to UniGene, GenBank and other Internet databases for sets of clones found to be of interest. Users may save their explorations on the Web server or local computer and later recall or share them with other scientists in this groupware Web environment. The emphasis on direct manipulation of clones and sets of clones in graphics and tables provides a high level of interaction with the data, making it easier for investigators to test ideas when looking for patterns. We have used the MAExplorer to profile gene expression patterns of 1500 duplicated genes isolated from mouse mammary tissue. We have identified genes that are preferentially expressed during pregnancy and during lactation. One gene we identified, carbonic anhydrase III, is highly expressed in mammary tissue from virgin and pregnant mice and in gene knock-out mice with underdeveloped mammary epithelium. Other genes, which include those encoding milk proteins, are preferentially expressed during lactation. MAExplorer may be accessed at http://www.lecb.ncifcrf.gov.MAExplorer.     

Physical mapping of yeast artificial chromosomes containing sequences from the human β-globin gene region

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human β-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult β-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire β-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the β-globin gene cluster.     

Isolation and propagation of yolk-sac-derived endothelial cells from a hypervascular transgenic mouse expressing a gain-of-functionFPS/FES proto-oncogene

Summary We report on the isolation and propagation of endothelial cells from the mouse embryonic yolk sac, the earliest site of blood vessel development, and on the advantages of a hypervascular transgenic mouse source of these cells. These transgenic mice express multiple copies of an activated allele of the humanfps/fes proto-oncogene and display hypervascularity progressing to multifocal hemangiomas. This phenotype suggested a role of thefps/fes proto-oncogene in vasculogenesis and angiogenesis and led us to investigate the growth characteristics of yolk-sac-derived endothelial cells from transgenicfps/fes embryos. We have established eight independent cell clones from a mixture of transgenic and control yolk sacs from Day 12 embryos. Southern blot hybridization analysis showed all eight clones to be derived from transgenic cells suggesting a growth advantage of cells carrying the activatedfps/fes gene. A cell line, Clone 166 (C166), established from one of these clones, was more fully characterized. C166 exhibits normal endothelial characteristics, such as rearrangement into tubelike structures when placed on Matrigel, expression of angiotensin converting enzyme, retention of cobblestone morphology at confluence, and the presence of cell surface receptors for acetylated low density lipoprotein. The cells constitutively express murine endothelial cell adhesion molecule VCAM-1 and the vascular addressin identified by antibody MECA-99. As expected, the cell line expresses high levels of the cytoplasmic protein-tyrosine kinase encoded by thefps/fes proto-oncogene. The clone we have described as well as other endothelial cell lines that we have established from the mouse embryonic yolk sac should prove useful for the study of endothelial cell differentiation and for the determination of the mechanisms underlying the establishment of organ-specific endothelial cell heterogeneity.     

Human adipose tissue is a source of multipotent stem cells

Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.     

Construction of a Tc1-like transposon Sleeping Beauty-based gene transfer plasmid vector for generation of stable transgenic mammalian cell clones

We have constructed a single plasmid-, Tc1-like transposon-based gene transfer vector, termed the Prince Charming vector (pPC). The pPC vector was constructed by ligating the CMV-driven "Sleeping Beauty" transposase gene downstream to the Tc1-like transposon inverted repeat (IR) elements and by inserting the RSV promoter (to drive expression of the gene-of-interest) along with a multiple cloning site (MCS), a polyadenylation signal, and the SV40 promoter-driven neomycin gene, at a site flanked by the transposon IR elements. To assess the utility of the pPC vector, we cloned a red fluorescent protein (RFP) gene into the pPC vector at the MCS and transfected human TE85 osteosarcoma cells with the pPC-RFP expression vector using Effectene. Stable transgenic cell clones expressing RFP were selected with G418 sulfate and individual clones were isolated. After 4 weeks of clonal isolation and expansion, 99% of cells in each randomly selected clone expressed RFP strongly. Aliquots of each clone were then maintained in either the presence or the absence of G418 sulfate and were passaged weekly. Even after 6 months in culture in the absence of G418 sulfate, approximately 90% of the cells in each clone still maintained a strong expression level of RFP, indicating that these transgenic cell clones were stable and that the clonal stability of these clones did not require a constant selection pressure. In conclusion, we have developed a single plasmid-, Tc1-like transposon-based gene transfer vector that can be used to generate stable transgenic mammalian cell clones.     

Utilization of non-AUG initiation codons in a flow cytometric method for efficient selection of recombinant cell lines

Here we describe a method that couples flow cytometric detection with the attenuated translation of a reporter protein to enable efficient selection of CHO clones producing high levels of recombinant proteins. In this system, a small cell surface reporter protein is expressed from an upstream open reading frame utilizing a non-AUG initiation (alternate start) codon. Due to the low translation initiation efficiency of this alternate start codon, the majority of translation initiation events occur at the first AUG of the downstream open reading frame encoding the recombinant protein of interest. While translation of the reporter is significantly reduced, the levels are sufficient for detection using flow cytometric methods and, in turn, predictive of protein expression from the gene of interest since both ORFs are translated from the same mRNA. Using this system, CHO cells have been sorted to obtain enriched pools producing significantly higher levels of recombinant proteins than the starting cell population and clones with significantly better productivity than those generated from limiting dilution cloning. This method also serves as an effective screening tool during clone expansion to enable resources to be focused solely on clones with both high and stable expression.     

Stochastic Fluctuations and Distributed Control of Gene Expression Impact Cellular Memory

Despite the stochastic noise that characterizes all cellular processes the cells are able to maintain and transmit to their daughter cells the stable level of gene expression. In order to better understand this phenomenon, we investigated the temporal dynamics of gene expression variation using a double reporter gene model. We compared cell clones with transgenes coding for highly stable mRNA and fluorescent proteins with clones expressing destabilized mRNA-s and proteins. Both types of clones displayed strong heterogeneity of reporter gene expression levels. However, cells expressing stable gene products produced daughter cells with similar level of reporter proteins, while in cell clones with short mRNA and protein half-lives the epigenetic memory of the gene expression level was completely suppressed. Computer simulations also confirmed the role of mRNA and protein stability in the conservation of constant gene expression levels over several cell generations. These data indicate that the conservation of a stable phenotype in a cellular lineage may largely depend on the slow turnover of mRNA-s and proteins.     

A modified procedure for replica plating of mammalian cells allowing selection of clones based on gene expression.

The polyester cloth replica-plating technique for selection of mammalian cell clones was modified by growing cells in colonies on a flexible polytetrafluoroethylene membrane and then transferring them completely to polyester cloth (27-microns mesh), from which a replica was made by allowing cells to transfer to a cloth of smaller pore size (17-microns mesh). Using this technique, two phenotype selection methods are demonstrated here: in situ hybridization for detection of a specific mRNA and a photographic film assay for detection of luciferase expression. Cells were transfected with pSV2AL-A delta 5' in which firefly luciferase cDNA is under the control of the simian virus 40 promoter. The luciferase assay was adapted for colonies on polyester cloth; cells were permeabilized with digitonin to allow access of ATP and luciferin to the cell without disruption of colonies. Clones selected for expression or nonexpression of luciferase by the photographic film assay were positive or negative for expression after isolation from the cloth replica and subsequent growth under conventional culture conditions. The replica-plating procedure described here should be generally applicable to most mammalian cell types. The ability to produce replicas of colonies, combined with in situ hybridization or assays that can be adapted to in situ detection, provides phenotype selection for clones based on gene expression independent of growth characteristics.     

Analysis of mammalian pigmentation at the molecular level

There has been great interest lately in the cloning of pigment-related genes; several laboratories have succeeded in isolating melanocyte-specific genes which have many of the characteristics expected for tyrosinase. In this paper, we review the selection criteria, the physical properties, and the functional characteristics of several of these gene products. Two of the clones map to the brown (b) and albino (c) loci, genes that are involved in the regulation of the quantity and quality of melanin production. The functional characteristics of these gene products are not easily reconciled with existing schemes of melanogenesis, and a reevaluation of our concepts of melanogenic regulation may be necessary. The altered expression of these gene products in normal and in transformed melanocytes, and the alternative mRNA processing that occurs in those cells, makes this system an appropriate and interesting one for studies of normal metabolic regulation of gene expression, as well as altered gene expression by neoplastic cells.     

A 'select and swap' strategy for the isolation of clones with tightly regulated transgenes.

Increasing numbers of biological problems are being addressed by genetic approaches that rely on inducible expression of transgenes. It is desirable that expression of such a transgene is tightly regulated, from close to zero expression in the 'off' state, to appreciable (at least physiological) expression in the 'on' state. Although there are many examples where tight regulation has been achieved, certain factors, including chromosomal position effects due to random integration of the transgene, often cause suboptimal inducibility and make the isolation of tightly regulated clones difficult and/or laborious. Here we describe a 'select and swap' strategy for the isolation, from a population of stable transfectants, of clones with tightly regulated transgenes. In this approach, a positively and negatively selectable, inducible marker gene is used to select for clones with optimal transgene regulation. After isolation of such clones, the marker gene is swapped with a linked gene of interest by the use of site-specific recombination. To test this strategy we introduced into human cells a plasmid with a tetracycline-inducible bacterial gpt gene linked to a promoterless luciferase gene, isolated clones with tight gpt expression and used the Cre/loxP site-specific recombination system to swap the gpt gene with the luciferase gene. We discuss ways for refining and developing the system and widening its applicability.     

Elevated frequency of microsatellite mutations in TK6 human lymphoblast clones selected for mutations at the thymidine kinase locus.

A major question in carcinogenesis is, How can a normal cell accumulate multiple mutations in different genes on different chromosomes, when the mutation rate of each gene is in the range of 10(-8) to 10(-5) per cell division? We hypothesize that many mutations may not be isolated events but rather are accompanied by concomitant mutations elsewhere in the genome. To test this hypothesis, 331 independent clones selected for new mutations at the thymidine kinase (TK) locus on chromosome 17q, and 243 nonselected control clones were examined for mutations in 12 random microsatellite loci dispersed throughout the genome. A total of 24 second-site mutations were identified in the TK mutant clones, compared with 3 in the control clones not selected for mutations at TK. The mutations include small deletions, insertions, and loss of heterozygosity. These results provide evidence that a global trans-acting mutagenic process exists in human cells. The activation of this process could be responsible for causing multiple essential mutations in tumor cells.