Premade cDNA Libraries

Suppliers of Whole-organism cDNA Libraries Suppliers of Tissue-specific cDNA Libraries The questions of gene function, interaction, and regulation are central to the science of molecular biology. Despite the myriad of new technologies, products, and techniques produced by the genomics revolution, some old standards remain just as useful as ever. One such technology is the cDNA (complementary DNA) library. The sheer number of companies offering premade and custom cDNA libraries and products that facilitate their use serves as evidence of the tool's sustained power and efficacy. Assessing which genes are active in a given tissue at a specific time, and determining the relative degree of gene activity are essential steps in understanding how the genome as a whole functions within the tissue or cell under investigation. The term "transcriptome" refers to the set of genes that are expressed or transcribed from genomic DNA in a particular cell or tissue. An understanding of the transcriptome translates into an understanding of the activity and roles of the proteins that these genes encode. For technical reasons however, it is much easier to work with DNA than with RNA. Therefore, researchers synthesize cDNA, which is a DNA copy of mRNA, in vitro. This produces a highly versatile tool that can be used to study gene regulation, expression, splice variation, post-translational modifications, and protein-protein interactions. Changes in patterns of gene expression provide valuable information about the causes and consequences of disease, the body's response to drugs, and the normal operation of cells. Unfortunately, the preparation of a cDNA library is a challenging, time-consuming, and complex process. The first step, extracting the cellular RNA, is itself a difficult task because of the prevalence of RNA-degrading enzymes (RNAses). Most functional mRNAs have a 3' poly-adenine tract, enabling researchers to purify the mRNA by passing total cellular RNA over a poly-thymine oligonucleotide column. The oligo-dT provides a 3'-hydroxyl group to prime first-strand synthesis with reverse transcriptase. This DNA strand usually turns back on itself to form a hairpin loop before the completion of synthesis, providing a primer for second strand synthesis with DNA polymerase I. An added RNAse H degrades the remaining mRNA, and SI nuclease opens the hairpins, degrading the extension. The resulting cDNA copies can be cloned by blunt-end ligation into a plasmid cloning vector to form a cDNA library, which can then be screened by DNA hybridization or by immunological assays. Researchers facing this grueling process often turn to commercially prepared libraries. Companies such as BD Biosciences-CLONTECH of Palo Alto, Calif., and Stratagene of La Jolla, Calif., offer researchers a large and diverse array of cDNA libraries, isolated from a striking variety of organisms and tissues. Many other companies offer more limited groups of libraries and all can produce customized libraries for researchers whose needs are not met through existing libraries. Working on Your Expressions cDNA libraries are commonly used in gene expression studies. Expression cloning of cDNAs using mammalian cells was first described more than a decade ago and has been used to isolate a variety of genes.1 The most common approach is to identify the cDNA clone of interest within the total population of cDNA clones by screening with a nucleic acid probe consisting of a previously cloned cDNA fragment, genomic DNA, or a synthetic oligonucleotide specifying a particular RNA sequence. Alternatively, cDNA expression libraries can be used to identify sequences encoding polypeptides or proteins of interest. Certain lambda vectors allow the expression of the polypeptide encoded by its DNA insert and can be screened with an antibody raised against the protein of interest. Nucleic acid-binding proteins can be cloned from expression libraries based on the ability to bind a labeled DNA or RNA fragment. For investigators interested in expression screening in mammalian cells, Carlsbad, Calif.-based Invitrogen's Discovery Line(tm) premade cDNA libraries are available prepared from human normal, fetal, or tumor tissue. The eukaryotic expression vector pcDNA3.1 is used to construct these libraries to facilitate expression in mammalian cells. These libraries are cloned unidirectionally (5' to 3') to reduce vector background and increase screening efficiency. The vector, which is also ideal for screening in Escherichia coli, is shipped ready to plate. The Discovery Line, offered through Invitrogen's subsidiary company ResGen of Huntsville, Ala., also includes Gene Pool(tm) cDNA, a variety of PCR-ready, first-strand cDNAs prepared using mRNA from human tumor, disease, fetal, and normal tissues. Gene Pool cDNAs are intended for the amplification of genes with specific or degenerate primers, analysis of tissue-specific expression, and characterization of alternately spliced mRNA. CLONTECH provides a broad selection of application-specific libraries that allow researchers to tailor their libraries to their specific research needs. CLONTECH's newest line of plasmid cDNA libraries are called Creator SMART(tm). Originally developed to serve as the foundation for the new Mammalian Gene Collection (MGC) project, a joint effort of the National Institutes of Health (NIH) and the National Cancer Institute (NCI), these libraries combine two proprietary technologies, Creator and SMART. cDNA is synthesized using SMART technology, which enriches the cDNA population with full-length genes by anchoring both the 3' and 5' ends of a gene using proprietary SMART oligonucleotides. Creator technology facilitates the easy movement of an insert from a single donor vector into a number of expression vectors using the Cre recombinase. As with any other plasmid or retroviral library, Creator SMART cDNA libraries can be screened by hybridization or PCR or with ClonCapture(tm) technology. ClonCapture mediates formation of stable complexes between library clones and a specific biotinylated probe. These complexes can be selected on streptavidin magnetic beads, thus enriching the library for specific sequences. CLONTECH's ClonCapture Libraries allow researchers to isolate clones based on a partial cDNA sequences. The libraries, which have been optimized for use with CLONTECH's ClonCapture Kit, are then available to be directly introduced into mammalian cells for expression studies. These kinds of libraries are intended for the identification of differentially spliced products and the isolation of clones that are homologous to a gene of interest. The ability to control transduction and gene expression is critical to functional studies with isolated cDNAs. To aid in controlling expression levels, Stratagene offers the ViraPort(tm) retroviral gene expression system, which allows for functional cloning of genes. Any researcher with a characterized functional assay can begin screening immediately using this technology. This system offers distinct advantages in cases where transfection efficiencies are low and controlling copy number is advantageous, or when cell types do not transfect well. The ViraPort retroviral gene expression system overcomes these limitations and provides efficient gene delivery using the optimized pFB vector backbone. Stratagene offers a number of human and murine libraries, available in two formats. The ViraPort VSV-G retroviral supernatants are ready for immediate transduction into a broad range of mammalian cell types including: mouse, rat, hamster, rabbit, mink, cow, cat, dog, human, and chicken. These supernatants can be used directly on virtually any target cell to infect and immediately conduct functional screens. Viral titers are provided with each library to allow the user to control copy number per cell, making identification of individual cDNA targets easier in one's functional screen. Alternatively, Stratagene offers a set of cDNA expression libraries inserted into pFB that are available as bacterial stocks. These libraries are ready for packaging in either a packaging cell line or by co-transfection with Stratagene's Vpack vectors. Finally, Stratagene offers a set of high-titer retroviral reporter vectors based on the pFB vector. The pFB-hrGFP vector contains Stratagene's exclusive variety of green fluorescent protein (GFP), pFB-neoLacZ uses the ß-galactosidase gene, and pFB-Luc expresses luciferase. Overcoming Problems The time it takes to accurately isolate and amplify clones of interest can be considerable, but the Library-in-a-Tube(tm) from Carlsbad, Calif.-based Qbiogene alleviates this problem. Library-in-a-Tube is a line of PCR-ready single-stranded cDNA libraries made from total RNA. Each library is offered in single-use 0.2-ml and 0.5-ml tubes, each of which contains a universal Taq buffer, dNTPs, and stabilizer for room temperature storage. Researchers can therefore amplify specific sequences or measure expression levels by simply adding primers and Taq polymerase. Each kit allows for three single-use PCR amplifications, with libraries available from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Bacillus subtilis, and E. coli. Another common problem is the identification of rare transcripts. According to Les Halpern, vice president of operations for Edge Biosystems of Gaithersburg, Md., researchers investigating less common transcripts benefit from using unamplified libraries: "Amplification induces bias. When you amplify a library, there is a tendency that the less common genes will not be well represented." Edge Biosystems offers an assortment of human and murine cDNA libraries, provided as either unamplified bacterial clones or as 1X amplified cDNA. One persistent challenge to researchers working with cDNA libraries is the isolation of full-length cDNAs. By offering vectors that incorporate larger inserts, and by incorporating long distance PCR (LD-PCR) into its cDNA library construction, CLONTECH is helping to mitigate this problem. Indeed, the company reports that cDNAs up to 9 kb in length have been cloned. CLONTECH's Large-Insert cDNA libraries have an average insert size of greater than 3 kb, twice that of the typical library, and are ideal for identifying full length clones. These libraries are constructed in the phagemid cDNA cloning vector, lambdaTriplEx2(tm), using LD-PCR to ensure a high representation of full-length cDNAs. CLONTECH also offers customized library screening services to assist researchers in locating full-length clones of genes of interest. Stratagene offers a system that addresses two concerns inherent in cDNA screening: time, and PCR-induced mutations. The sequence information generated from the Human Genome Project has made it possible to synthesize oligonucleotides for library screening by in situ hybridization or for direct PCR amplification from different RNA sources (RT-PCR). However, the screening process can be tedious and time-consuming, and RT-PCR can result in mutation, deletion, or insertion in the cDNA. To curb these obstacles, researchers can make use of Stratagene's Cloning Matrix(tm) system for its Human Universal cDNA Library (HUCL), which represents genes expressed in 29 different human tissues. These arrays, which have been used to isolate full-length genes that correspond to rare and long transcripts,2 allow rapid PCR screening while maintaining the fidelity of direct colony screening. The end product is a cDNA clone that was individually selected and cultured rather than a PCR product. The average insert size in the libraries is 1.7 kb, and 50 percent of the genes are estimated to be full-length. According to product manager Matthew Klimjack, "What we found was that customers wanted a PCR-based format for screening. Thus, we developed the Cloning Matrix System for HUCL. This allows the user to screen the entire HUCL library by setting up PCR reactions from our master 96-well plate ... and when compared to other methods of screening, the Cloning Matrix System is faster, easier, and uses no radioactivity. It's also a more economical system." The Cloning Matrix System master plate contains enough DNA for 10 complete screenings. Finally, for investigators with broad-scale interests such as apoptosis, drug interaction, or cancer, tissue-specific libraries are not always optimal. DNA Technologies (www.dnati.com) of Gaithersburg, Md., offers the Mouse Smart-cDNA(tm) Library Array, a universal cDNA library from a pool of various mouse tissues including liver, brain, heart, kidney, testis, and ovary, arrayed onto nylon membranes. The array includes seven membranes totaling approximately 500,000 clones. DNA Technologies also offers customized cDNA library and screening services. These libraries are prepared by fractionation of the cDNA into a small (0.5 to 2 kb) or a large fraction (2 kb and larger), thus facilitating the identification of the gene of interest. Social Interactions The two-hybrid system in yeast has become the method of choice to detect protein-protein interactions (recently profiled in The Scientist3). The technique can be used to test for interactions between predefined polypeptides, but it can also be used to screen complex libraries of genomic or complementary DNA for interacting partners. In the latter strategy, complex libraries of protein domains can be screened to saturation with high-throughput screening systems allowing the discovery of proteins.4 Stratagene's CytoTrap(r) two-hybrid system allows investigators to detect protein-protein interactions in the yeast cytoplasm, and provides a good way to study transcriptional regulators or proteins that depend on post-translational modifications in the cytoplasm. In addition to the vector kit and the library construction kit, Stratagene also offers CytoTrap premade libraries from a variety of human, bovine, Drosophila, and murine tissues. They are cloned into the pMyrXR vector and are provided complete with bait vector, yeast strains, and control plasmids. Insert sizes of the cDNAs range from 1.2 to 1.7 kb to ensure a high representation of clones in the library. The company's HybriZAP two-hybrid cDNA libraries, like most commercially available yeast two-hybrid libraries, are based on transcriptional activation but contain a unique ZAP vector that allows easy and rapid excision from the lambda phage into a plasmid format. The T7Select(r) line of cDNA libraries, produced by Novagen Inc. of Madison, Wis., is particularly useful for expression cloning and protein-protein interaction studies via phage display. In phage display analyses, a library of cDNAs is cloned in-frame with a viral coat protein, thus allowing the cloned gene to be expressed on the surface of the virus. Interacting proteins can then be selected via a "bio-panning" process. Novagen offers three different phage display vectors that differ based on the expression level of the cloned gene. The company's premade cDNA libraries are cloned in the T7Select10-3 mid-level expression vector, and libraries are available for a variety of normal and diseased human tissues. Novagen also offers a wide selection of pre-made lambda libraries. Microarrays vs. cDNA Libraries High-throughput, information-rich, broad-scale investigations are replacing the classical reductionistic molecular biology experiment. Thus, many scientists are replacing single gene discovery or sequencing experiments with microarray experiments. These high-density, solid-state arrays of oligonucleotides or cDNAs are probed by hybridization with labeled RNA or DNA in solution. The arrayed DNA fragments can be obtained from genomic, plasmid, or cDNA libraries, and arrays with more than 10,000 cDNAs per square centimeter are available and provide an incredibly powerful tool, particularly for comparative expression studies. One such study discovered 289 genes whose expression patterns differed significantly when normal colon epithelial cells were compared with colon cancer cells; about half of these genes also exhibited abnormal expression levels in pancreatic cancer cells.5 Some scientists envision a decrease in the popularity of libraries. According to Mary Wieder of Invitrogen technical services, "While the cDNA libraries will continue to be used in certain labs...many scientists are finding that arrays, SAGE [serial analysis of gene expression], and other more creative high-throughput techniques serve their purposes more effectively in terms of time and cost." Others, such as Stratagene's Klimjack, counter that "there is still enthusiasm for cDNA libraries [and] definitely a high degree of interest in plasmid libraries for functional cloning." In many instances, cDNA libraries may be used in microarray experiments, providing the DNA to be spotted and hybridized in expression studies. While the introduction of array technology makes it possible to monitor the expression of thousands of genes in single experiments,6 the approach is limited. The frequency of a gene expressed in a cDNA library, as determined by techniques such as SAGE and expressed sequence tag (EST) counts, is a measure of its tissue-specific expression and an approach that is complementary to data produced by microarray expriments.7 Of course, the sheer volume of information produced by the use of multiple cDNA libraries can be overwhelming. In 1993, four academic groups collaborated to form the Integrated Molecular Analysis of Genomes and their Expression (I.M.A.G.E.) Consortium (image.llnl.gov). The goal of the group was to array and share high-quality cDNA libraries and to make sequence and expression data about these clones available to any interested researcher via the Internet. The Consortium, which works with oligo- dT-primed, directionally cloned plasmid cDNA libraries, has arrayed clones from human, mouse, rat, zebrafish, and Xenopus libraries. I.M.A.G.E. consortium clones are available royalty-free, and may be obtained for a nominal fee (usually around $30). Likewise, libraries can be purchased for a small fee from any of the consortiums five distributors: Manassas, Va.-based American Type Culture Collection (www.atcc.org), Palo Alto, Calif.-based Incyte Genomics (www.incyte.com), Invitrogen's ResGen subsidiary, the Hinxton, England-based UK Human Genome Mapping Project Resource Centre (www.hgmp.mrc.ac.uk), and the Berlin-based Resource Center of the German Human Genome Project (www.rzpd.de). Sarah Goforth (SGoforth@molbio.princeton.edu) is a freelance writer in Princeton, N.J. References 1. U. Gubler, B.J. Hoffman, "A simple and very efficient method for generating cDNA libraries," Gene, 25:263-9, 1983. 2. A. Mehta et al., "Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA," Molecular and Cell Biology, 20:1846-54, March, 2000. 3. B.A. Cunningham, "Finding a mate," The Scientist, 15[7]:26, April 2, 2001. 4. P. Legrain, L. Selig, "Genome-wide protein interaction maps using two-hybrid systems," FEBS Letters, 480:32-6, Aug. 25, 2000. 5. L. Zhang et al., "Gene expression profiles in normal and cancer cells," Science, 276:1268-72, 1997. 6. B. Phimister et al., "Chipping forecast," Nature Genetics (supplement), 21:1-60, 1999. 7. D.J. Stekel et al., "The comparison of gene expression from multiple cDNA libraries," Genome Research, 10:2055-61, Dec., 2000.

Premade cDNA Libraries

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