Use Of Mammalian Cells Research Articles

Genetic Manipulation of Mammalian Cells by Microinjection: Figure 1.

INTRODUCTIONMicroinjection allows the introduction of molecules into a defined population of cells at a defined concentration, and the timing of the experiment can be controlled stringently, minimizing problems associated with overexpression. Delivery by microinjection can be used for any type of cell that is adherent in culture, including primary cells. Perhaps its most powerful aspect is the ability to introduce several types of reagents into cells simultaneously, including DNA constructs, a labeled dextran to mark injected cells, antibodies, short interfering RNAs (siRNAs), and peptides. Because siRNA can be generated easily and rapidly for any target gene, it is relatively simple to assess many effects of the knockout of any gene in any type of adherent cell in a matter of days. However, microinjection is not trivial to learn, and requires attention to detail. Also, the limited number of cells involved often does not permit the subsequent analysis of effects on some biochemical parameters. While the major limitation of the approach is the small amount of material obtained, as the sensitivity of analysis techniques increases, so does the usefulness of this method. We emphasize here recent advances in the use of microinjection in combination with siRNA technology.

An optimized protocol for protein purification in cultured mammalian cells using a tandem affinity purification approach

This protocol describes a method that we developed to adapt the tandem affinity purification (TAP) approach for use in mammalian cells. The protocol involves fusing a protein of interest with a tandem tag consisting of two FLAG tags (FF) followed by two protein-A immunoglobulin G (IgG) binding domains (ZZ). The protocol improves upon previously published TAP approaches by employing FLAG in place of calmodulin binding peptide (CBP) with resulting higher recovery during purification. In addition, we use a bicistronic expression system that ensures recovery of stably transfected cell lines expressing easily detectable levels of the protein of interest. A method is also presented for generating cytoplasmic and nuclear extracts, which extends use of this protocol to identify protein-protein interactions occurring specifically in the cytoplasm or nucleus. This protocol facilitates the preparation of partially purified recombinant protein and identification of protein-protein interactions in mammalian cell culture models. The protocol can be completed in 34 h.

An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol that enables the isolation of protein complexes under close-to-physiological conditions for subsequent analysis by mass spectrometry. Although TAP was instrumental in elucidating the yeast cellular machinery, in mammalian cells the method suffers from a low overall yield. We designed several dual-affinity tags optimized for use in mammalian cells and compared the efficiency of each tag to the conventional TAP tag. A tag based on protein G and the streptavidin-binding peptide (GS-TAP) resulted in a tenfold increase in protein-complex yield and improved the specificity of the procedure. This allows purification of protein complexes that were hitherto not amenable to TAP and use of less starting material, leading to higher success rates and enabling systematic interaction proteomics projects. Using the well-characterized Ku70-Ku80 protein complex as an example, we identified both core elements as well as new candidate effectors.

A model of oncogenic rearrangements: differences between chromosomal translocation mechanisms and simple double-strand break repair

AbstractRecurrent reciprocal translocations are present in many hematologic and mesenchymal malignancies. Because significant sequence homology is absent from translocation breakpoint junctions, non-homologous end-joining (NHEJ) pathways of DNA repair are presumed to catalyze their formation. We developed translocation reporters for use in mammalian cells from which NHEJ events can be selected after precise chromosomal breakage. Translocations were efficiently recovered with these reporters using mouse cells, and their breakpoint junctions recapitulated findings from oncogenic translocations. Small deletions and microhomology were present in most junctions; insertions and more complex events also were observed. Thus, our reporters model features of oncogenic rearrangements in human cancer cells. A homologous sequence at a distance from the break site affected the translocation junction without substantially altering translocation frequency. Interestingly, in a direct comparison, the spectrum of translocation breakpoint junctions differed from junctions derived from repair at a single chromosomal break, providing mechanistic insight into translocation formation. (Blood. 2006;107:777-780)

Genome‐wide screening for gene function using RNAi in mammalian cells

Mammalian genome sequencing has identified numerous genes requiring functional annotation. The discovery that dsRNA can direct gene-specific silencing in both model organisms and mammalian cells through RNA interference (RNAi) has provided a platform for dissecting the function of independent genes. The generation of large-scale RNAi libraries targeting all predicted genes within mouse, rat and human cells, combined with the large number of cell-based assays, provides a unique opportunity to perform high-throughput genetics in these complex cell systems. Many different formats exist for the generation of genome-wide RNAi libraries for use in mammalian cells. Furthermore, the use of these libraries in either genetic screens or genetic selections allows for the identification of known and novel genes involved in complex cellular phenotypes and biological processes, some of which underpin human disease. In this review, we examine genome-wide RNAi libraries used in model organisms and mammalian cells and provide examples of how these information rich reagents can be used for determining gene function, discovering novel therapeutic targets and dissecting signalling pathways, cellular processes and complex phenotypes.

Towards the In Vivo Identificaton of Leukemogenic Fusion Proteins.

Currently there are no methods available to identifiy leukemogenic fusion proteins in vivo. All available methods, like Southern blotting, PCR, FISH or Western blotting, require the destruction of the cells that are assayed. A method for the in vivo detection of leukemogenic fusion proteins would be highly desirable because it would open up new approaches to study leukemia and might lead to novel treatment strategies.We have developed a strategy for the in vivo detection of the BCR/ABL fusion protein. BCR/ABL is found in virtually all cases chronic myeloid leukemia (CML) and a large proportion of acute lymphoblastic leukemia (ALL). Animal model have shown that the BCR/ABL fusion protein is required for the induction and maintenance of leukemia. The fact that BCR/ABL fusion protein is crucial for the development of leukemia makes this fusion protein an attractive target for therapy development. Our BCR/ABL detection strategy is based on protein-protein interactions and a proof of principle for the strategy was implemented in the yeast system. Two detection proteins are expressed in the cells: 1) protein A, a Gal4-DNA binding domain/BCR interacting protein fusion protein and 2) protein B, a Gal4-activation domain/ABL interacting protein fusion protein. Only when BCR/ABL is present in the cell, do protein A, protein B, and BCR/ABL form a trimeric complex which activates the transcription of reporter genes under the control of Gal4-upstream activating sequence (UAS).Yeast cells (strain CG1945) transformed with a protein A expressing plasmid (pGBT9-BCR-interactor), a protein B expressing plasmid (pGAD424-ABL1-interactor), and a BCR/ABL expressing plasmid (pES1-BCR/ABL) showed expression of the reporter genes HIS3 and LACZ. The expression of the HIS3 reporter gene was assayed by growth of the yeast cells on medium lacking histidine. The expression of the LACZ gene was verified by a beta-galactosidase filter assay.Yeast cells that were transformed with the pES1 plasmid without the BCR-ABL coding region did not show activation of the reporter genes. Several other negative controls were also negative. Thus the method was able to clearly distinguish between BCR/ABL expressing cells and cells did not express BCR/ABL.We are presently adapting this system for use in mammalian cells. The flexibility of our strategy allows us to freely choose the reporter or effector genes. Therapeutically more useful effector genes are suicide genes, which encode pro-drug converting enzymes (e.g. HSV thymidine kinase), or markers that can easily be assayed (e.g. green fluorescent protein).

Modification of HPV 16 E7 genes: correlation between the level of protein expression and CTL response after immunization of C57BL/6 mice

Immunization with a codon-optimized HPV 16 E7 gene was shown to yield higher levels of E7-specific cytotoxic T cells [Liu WJ, Gao F, Zhao KN, Zhao W, Fernando GJ, Thomas R, et al. Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity. Virology 2002;301:43]. Here, we sought to verify the hypothesis that there is a direct correlation between the level of protein expression and immunogenicity in mice. We generated HPV 16 E7 expression plasmids where the genes were inserted either as authentic sequence (wt) or after optimizing the codons for use in mammalian cells (opt). For enhancement of translation of the E7 gene a 5′ Kozak sequence (K) was added. Transfection experiments revealed the strength of expression in the order of E7opt + K, E7opt − K, E7wt + K and E7wt − K. After immunization of C57/B6 mice we observed an equally strong CD8 + T-cell response with the E7opt plasmids (+ or −K), followed by the E7wt + K and E7wt − K DNAs. The same difference in efficiency was obtained in tumor protection experiments. Regression of pre-existing tumors and CTL activity was observed only with the E7opt + K plasmid. From these data, we conclude that the level of protein expression correlates with the efficiency of CTL response and hence testing by transfection of cells in culture may allow a pre-selection of expression plasmids prior to DNA immunization.

Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells.

An orthogonal tryptophanyl-transfer RNA (tRNA) synthetase (TrpRS)-mutant opal suppressor tRNA(Trp) (mutRNA(UCA)(Trp)) pair was generated for use in mammalian cells. The anticodon loop of the Bacillus subtilis tRNA(Trp) was mutated to UCA, three positions in the D arm were mutated to generate an internal promoter sequence, and the mutRNA(UCA)(Trp) gene was inserted between the 5' and 3' flanking sequences of the tRNA(Trp-1) gene from Arabidopsis to enhance its expression in mammalian cells. In vitro aminoacylation assays and in vivo opal suppression assays showed that B. subtilis TrpRS (BsTrpRS) charges only the cognate mutRNA(UCA)(Trp) and no endogenous mammalian tRNAs. Similarly, the mutRNA(UCA)(Trp) is specifically charged by B. subtilis TrpRS and not by endogenous synthetases in mammalian cells. Site-directed mutagenesis was then used to alter the specificity of BsTrpRS to uniquely charge 5-hydoxy-l-tryptophan. The resulting mutant BsTrpRS-mutRNA(UCA)(Trp) pair allows the efficient and selective incorporation of 5-hydroxy-l-tryptophan into mammalian proteins in response to the codon, TGA. This amino acid can be used as a fluorescence probe and also undergoes electrochemical oxidation in situ to generate an efficient protein crosslinking.

Exploiting features of adenovirus replication to support mammalian kinase production.

Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated.

Modulation of Gene Expression by Lentiviral-Mediated Delivery of Small Interfering RNA

RNA interference (RNAi) mediates sequence-specific posttranscriptional gene silencing triggered by double-stranded RNA. RNAi is an effective tool for functional genomics in C. elegans, whereas biochemical characteristics of RNAi hamper its use in mammalian cells. We here analyze kinetic and quantitative aspects of mammalian RNAi in different cell lines using novel lentiviral constructs with double H1-shRNA expression cassettes located in the U3 region of the LTR. Using enhanced green fluorescence protein (EGFP) as a target gene for RNAi and red fluorescence protein (RFP) as surrogate marker for intracellular siRNA expression, we show that long-term siRNA expression mediates stable RNAi. Furthermore, RNAi-induced gene silencing varies from minimal to complete loss-of-function phenotypes within homogenous and between different cell populations. Interestingly, the extent of gene silencing correlates to lentiviral integrations as well as siRNA expression levels in target cells. Finally, we demonstrate functional gene silencing of a cytokine receptor gene in normal CD34+ hematopoietic progenitor cells. These data suggest that the use of suitable lentiviral constructs to quantify siRNA expression in living cells may allow analysis of loss-of-function phenotypes in a dose-dependent manner in a wide variety of target cells.

Efficient interference

Although double-stranded RNA-induced gene silencing (or RNA interference, RNAi) has proven to be a useful tool for genetic manipulation in a wide range of organisms, its use in mammalian cells has been very limited until recently. More than a year ago, Tuschl and colleagues reported the effective use of short interfering RNAs (siRNAs) in mammalian cells, possibly by bypassing the antiviral response of cells usually triggered by dsRNA.

Application of recombinant fluorescent mammalian cells as a toxicity biosensor

With respect to developing a more sensitive biosensor, a recombinant fluorescent Chinese Hamster Ovary cell line was used for the monitoring of various toxicants. Both cell lines, EFC-500 and KFC-A10, were able to detect toxicants sensitively. They were characterized with mitomycin C and gamma-ray as genotoxicants and bisphenol A, nonylphenol, ziram and methyl bromide as possible and known EDCs. When compared to each other, the response of KFC-A10 was generally more informative and sensitive. Compared to typical bacterial biosensor systems, these cell lines offered a sensitivity of 2- to 50-fold greater for the tested chemicals. Based on these results, the use of mammalian cells offers a sensitive biosensor system that is not only fast, cheap and reproducible but also capable of monitoring the endocrine-like characteristics of environmental toxicants.

Isolation of antigen-specific intracellular antibody fragments as single chain Fv for use in mammalian cells.

Isolation of antigen-specific intracellular antibody fragments as single chain Fv for use in mammalian cells.

Cellular biosensors for drug discovery

Recent advances in cell biology, fluorescent probe chemistry, miniaturization and automation have allowed the use of mammalian cells in a variety of medical and industrial applications. Here we describe the generation of cell-based biosensors, engineered to optically report specific biological activity. Cellular biosensors are comprised of living cells and can be used in various applications, including screening chemical libraries for drug discovery and environmental sensing. Panels of biosensors may also be useful for elucidating the function of novel genes. Here we describe two examples of the construction and use of engineered cell lines as biosensors for drug discovery.

Recombinant Semliki Forest virus for over-expression and pharmacological characterisation of the histamine H 2 receptor in mammalian cells

We describe the use of recombinant Semliki Forest virus (SFV) vectors for efficient expression of the rat histamine H 2 (rH 2) receptor in COS-7 (African green monkey kidney cells) cells. Recombinant SFV-infected COS-7 cells express the histamine rH 2 receptor in a time-dependent fashion with a maximum expression level of 50 pmol mg −1 after 40 h. SFV-mediated histamine rH 2 receptor expression shows similar pharmacological properties as the receptor expressed transiently or stably in mammalian cells. In addition, we demonstrate the pharmacological and functional characterisation of the D 115N mutated histamine rH 2 receptor. It has been shown that the D 115N mutation renders the receptor constitutively active and structurally unstable. The rapid onset of and high maximal expression levels obtained from SFV-infected COS-7 cells enabled us to characterise this mutant receptor. We prove that recombinant SFV vectors are powerful tools for heterologous expression of G-protein-coupled receptors and that one can achieve both the high-level gene expression described for baculovirus-infected insect cells and the use of mammalian cells as hosts.

TAT-Mediated Protein Transduction into Mammalian Cells

Manipulation of mammalian cells has been achieved by the transfection of expression vectors, microinjection, or diffusion of peptidyl mimetics. While these approaches have been somewhat successful, the classic manipulation methods are not easily regulated and can be laborious. One approach to circumvent these problems is the use of HIV TAT-mediated protein transduction. Although this technology was originally described in 1988, few improvements were reported in the subsequent 10 years. In the last few years, significant steps have been taken to advance this technology into a broadly applicable method that allows for the rapid introduction of full-length proteins into primary and transformed cells. The technology requires the synthesis of a fusion protein, linking the TAT transduction domain to the molecule of interest using a bacterial expression vector, followed by the purification of this fusion protein under either soluble or denaturing conditions. The purified fusion protein can be directly added to mammalian cell culture or injected in vivo into mice. Protein transduction occurs in a concentration-dependent manner, achieving maximum intracellular concentrations in less than 5 min, with nearly equal intracellular concentrations between all cells in the transduced population. Full-length TAT fusion proteins have been used to address a number of biological questions, relating to cell cycle progression, apoptosis, and cellular architecture. Described here are the fundamental requirements for the creation, isolation, and utilization of TAT-fusion proteins to affect mammalian cells. A detailed protocol for production and transduction of TAT-Cdc42 into primary cells is given to illustrate the technique.

Towards integrating vectors for gene therapy: expression of functional bacteriophage MuA and MuB proteins in mammalian cells.

Bacteriophage Mu has one of the best studied, most efficient and largest transposition machineries of the prokaryotic world. To harness this attractive integration machinery for use in mammalian cells, we cloned the coding sequences of the phage factors MuA and MuB in a eukaryotic expression cassette and fused them to a FLAG epitope and a SV40-derived nuclear localization signal. We demonstrate that these N-terminal extensions were sufficient to target the Mu proteins to the nucleus, while their function in Escherichia coli was not impeded. In vivo transposition in mammalian cells was analysed by co-transfection of the MuA and MuB expression vectors with a donor construct, which contained a miniMu transposon carrying a Hygromycin-resistance marker (Hyg(R)). In all co-transfections, a significant but moderate (up to 2.7-fold) increase in Hyg(R) colonies was obtained if compared with control experiments in which the MuA vector was omitted. To study whether the increased efficiency was the result of bona fide Mu transposition, integrated vector copies were cloned from 43 monoclonal and one polyclonal cell lines. However, in none of these clones, the junction between the vector and the chromosomal DNA was localized precisely at the border of the Att sites. From our data we conclude that expression of MuA and MuB increases the integration of miniMu vectors in mammalian cells, but that this increase is not the result of bona fide Mu-induced transposition.

Cre recombinase mediated alterations of the mouse genome using embryonic stem cells.

The introduction and establishment of transgenic, and in particular embryonic stem (ES) cell-based gene “knockout” technologies have made the mouse a key player in studying embryonic development and disease (1,2). In recent years, methods for the production of more complex genomic alterations have become increasingly widespread, hinting at an ability to manipulate and study a mammalian genome to an extent never previously thought possible. Such methodologies often partner homologous recombination-mediated gene targeting or random integration with site-specific recombination events. This chapter is concerned with the utilization of the bacteriophage P1 derived site-specific recombinase protein Cre (3–5), and its employment as a means to catalyze modifications in homologously recombined and randomly integrated target sites within the mouse genome. Cre is a 38-kDa protein that recombines DNA between two loxP target sites. loxP sequences are 34 basepairs (bp) long comprising two 13-bp inverted repeats flanking an asymmetric 8 bp core sequence. The recombination between two loxP sites with same orientation on the same DNA leaves two products each containing a single loxP site (6) (Fig. 1). This type of site-specific recombination, of which there are several other well-characterized systems in addition to the Cre/loxP, generates precise rearrangements of DNA but dispenses with the requirement for extensive homology between DNA partaking in the recombination. Recombination occurs through the recognition of the target sites by the recombinase, which then catalyzes strand exchange between them by precise breakage and rejoining events that are restricted to an internal region of identical sequence contained within the specific sites (6). Fig. 1 The Cre recombinase has a 34-bp recognition site comprising two inverted repeats and a core sequence (A). It can catalyze a site-specific recombination event leading to the deletion of the intervening DNA (B). In addition to the Cre/loxP system, another one of the many recombinase that does not require cis-elements, but utilizes short recognition sites for recombination is the yeast FLP/FRT system. This system has also been widely used and applied to genome alterations (7–10), though to date the Cre protein has been shown to be more amenable to use in mammalian cells, and is therefore currently favored by most laboratories for use in ES cells and transgenic mice (11). As a consequence, we will solely refer to the Cre/loxP system in the strategies we present, though it should be noted that if the FLP/FRT were to be as efficient as the Cre/loxP system it could be substituted in all methodologies. Additionally, it might also be anticipated that future experiments may require a multistep, site-specific recombination strategy, thereby requiring the use of two separate recombination systems. This chapter will illustrate and provide the methodologies for some of the applications of such a site-specific recombination system to experiments aimed at analyzing mouse embryonic development and/or disease conditions, from single-gene alterations, lineage-restricted and/or conditional gene ablations or ectopic expression through to chromosome engineering, and finally the use of such a system for lineage analysis. 1.1. Combining Homologous and Site-Specific Recombination Homologous recombination in ES cells allows the precise disruption (knockout) of a target gene. Many new approaches require a defined alteration of a gene or the genome. By combining the homologous and site-specific recombinations, we are now in the position of creating most desired alterations in the mouse genome (12,13). In the following section, we will briefly introduce some of the most important current applications. The list will not be complete, since novel applications for the use of this system are continuously being reported. 1.1.1. Eliminiating Any Regional Effect of a Knockout: Removal of a Selectable Marker To identify targeted events, an introduction of a positive selectable marker, usually neomycin, into the targeted locus is required. Recently, there has been an increasing concern regarding the repressor effect of the selectable marker cassette on the genes in the vicinity of its insertion. Therefore, removal of the marker from all targeted genes is advisable. This can most easily be performed by flanking (floxing[flanking with loxP]) the selectable marker cassette by loxP sites, which on introduction of the Cre recombinase will result in the removal of neomycin.

Improvement of mammalian cell survival by apoptosis-inhibiting genes and caspase inhibitors for effective use of mammalian cells: Satoshi Terada, Tsuyoshi Yata, Katsuyuki Fukuoka, Hiroshi Aisawa, Tetsuo Fujita, Teruyuki Nagamune, and Eiji Suzuki ( Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-86561 and Research Institute of Innovative Technology for the Earth,

Improvement of mammalian cell survival by apoptosis-inhibiting genes and caspase inhibitors for effective use of mammalian cells: Satoshi Terada, Tsuyoshi Yata, Katsuyuki Fukuoka, Hiroshi Aisawa, Tetsuo Fujita, Teruyuki Nagamune, and Eiji Suzuki ( Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-86561 and Research Institute of Innovative Technology for the Earth,

A new set of positive/negative selectable markers for mammalian cells.

Five new positive and negative selectable markers were created for use in mammalian cells. Their negative selectabilities are based on the Thymidine kinase (Tk) gene of Herpes Simplex virus (HSV) or the Cytidine deaminase (codA) gene of E. coli. The markers can be selected positively by their ability to induce either Hygromycin (Hyg), neomycin (neo), puromycin (PAC) or Blasticidin S (BlaS) resistance. With these markers, two complete sets of markergenes are available that induce independent negative selectable phenotypes.