Briefings in Functional Genomics and Proteomics Advance Access originally published online on November 19, 2007
Briefings in Functional Genomics and Proteomics 2007 6(3):171-179; doi:10.1093/bfgp/elm024
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Special Issue Papers |
Transcriptomics resources for functional genomics
Corresponding author. Yoshihide Hayashizaki, Genome Exploration Research Group, Genomic Sciences Center (GSC), RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. E-mail: yoshide{at}gsc.riken.jp
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ABSTRACT |
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TOP
ABSTRACT
IMPORTANCE OF FULL-LENGTH CDNA...For the past decade, extensive efforts have been made for establishing cDNA clone resources for various species. The most striking breakthrough has been full-length cDNA technology allowing cloning of intact RNA molecules as cDNA. These transcriptomics resources are providing researchers with essential tools for studies of functional genomics. Here, the importance of quality and availability of these resources is discussed.
Keywords: transcriptome, clone resource, FL-cDNA, ORF clones, DNA Book
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IMPORTANCE OF FULL-LENGTH CDNA (FL-CDNA) CLONE RESOURCES FOR FUNCTIONAL GENOMICS |
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Recent rapid progress in the analysis of genomes and transcriptomes of a variety of species, including human, has dramatically accelerated functional genomics research. One of the challenging approaches in functional genomics is to elucidate the gene networks or gene interactions, which underlie various biological phenomena such as cell growth/differentiation, immune reaction, oncogenesis and other diseases. Currently emerging trends of post-genome research require systematic analysis of molecules in the cell in a genome-wide manner. For example, in the Genome Network Project (GNP) in Japan, around 30 institutes are exploring the network of genes involved in transcriptional regulation in human (http://genomenetwork.nig.ac.jp/index_e.html). RIKEN (a core institute of the project) is conducting comprehensive screening of protein–protein interaction, especially of transcriptional factors, by using a mammalian two-hybrid assay [1, 2]. Therein, protein-coding regions amplified from transcriptional factor cDNAs are transfected with a candidate gene, and the binding activities of two proteins translated from cDNAs are assayed using the fluorescent level corresponding to reporter gene expression. Thus, full-length cDNA clone resources which allow researchers to syntesize proteins are absolutely essential. Genomic DNA clones are not generally suitable for functional genomics, since final proteins can only be obtained from cDNA clones. Here, we introduce various cDNA clone resources available for functional genomics and argue for the importance of efforts for maintaining good qualities of cDNA clones. We will also argue for continuous efforts to collect missing cDNA clones including non-protein coding RNAs for future functional genomics.
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ESTABLISHMENTS OF PRIMARY FL-cDNA CLONE RESOURCES |
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In the past decade, many cDNA projects for several species have contributed to establish the cDNA clone resources (Table 1). At the initial stages, cDNA projects have been aimed at collecting as many cDNA clones as possible in full-length form, despite the diversity in transcript lengths and expression. Thus, high efficiency of cDNA cloning has been the biggest problem, since long mRNA molecules are generally hard to convert into cDNA by reverse transcriptase (RT), and rarely expressed mRNAs are often missed. To address this problem, various technologies were developed, for example, wide-range cDNA cloning vectors with sizes ranging from a hundred bases to over 15 kb [20], thermo-stable RT reaction [21] and normalization/subtraction methods for enriching novel transcripts [22].
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Mammalian FL-cDNA clone resources
The most comprehensive FL-cDNA clone set for mammals is the mouse FANTOM clone set established in the Riken Mouse Genome Encyclopedia Project. So far, over 2 million of FL-cDNA clones have been generated end-sequenced either or both from the 3'- and 5'- end. After clustering these end-sequences, full sequences representing 103 000 clones were determined [6–8]. These full-length clones were synthesized from 246 tissue and cell samples including embryos in the early developmental stage and rare immune cells fractionated by the cell sorter. Two million clones had been picked up after subtraction by pre-collected clones as probes, in order to avoid duplicated cloning of the same cDNAs, suggesting that it could represent more than12 million mRNA molecules [23, 24]. The FANTOM mouse FL-cDNA clone set is, thereby, now the most comprehensive transcriptome resource in mammals. Furthermore, the FANTOM clones have several features: (i) A high rate of full-lengthness covering whole protein-coding regions. Over 95% of clones are of full-length at the FL-cDNA library construction step, and over 60% of them are of full-length at the final full-sequencing step, although this estimation of the full-lengthness rate might be slightly incorrect due to the existence of non-coding RNA. (ii) The functional annotation of each gene is available (http://fantom3.gsc.riken.jp/), curated by researchers for every gene. (iii) By re-sequencing of each of the clones, the correct correspondence between the clones and the sequence information in public DNA database are confirmed.
For human FL-cDNA clones, the Mammalian Gene Collection (MGC) was extensively conducted by NIH. At present, 25 853 FL-cDNA clones are available from various distributors for functional research (http://mgcc.nci.nih.gov) [9, 25, 26]. In contrast with the FANTOM clones, the MGC project focuses only on full-length sequencing of the cDNA clones that appear to encode for proteins and disregard non-coding RNA. Also, FLJ clones (full length, Japan) developed by the Tokyo University (http://www.nedo.go.jp/bio-e/) [4] and KIAA clones developed by the Kazusa DNA Research Institute (http://www.kazusa.or.jp/huge/) [5] are available. Notably, the KIAA clones are focused on large transcripts longer than 5 kb. It provides excellent tools, since longer transcripts such as of ion channels and extra-cellular matrix proteins are generally difficult to clone.
Plant FL-cDNA clone resources
FL-cDNA projects of two model plants, rice for monocot plants and Arabidopsis for dicotyledonous plants, have been carried out [14–16]. As for rice, 28 469 FL-cDNA clones together with their full-sequences are available from the National Institute of Agrobiological Sciences (NIAS) (http://cdna01.dna.affrc.go.jp/cDNA/). This collection includes 19 000–20 500 species of transcripts and covers 76% of the predicted protein-coding transcripts. From Arabidopsis, 224 336 FL-cDNA clones with 3'-end sequences have been released by RIKEN (http://rarge.psc.riken.jp/), out of which 15 295 clones were entirely sequenced. Genome mapping of cDNA sequences indicates that these clones can be classified into 18 090 distinct kinds of transcripts, which corresponds to about half of the 26 828 Arabidopsis genes annotated for the genome [14, 15]. RIKEN FL-cDNA technology contributed to these projects for establishing the high-quality full-length cDNA clones.
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cDNA CLONE RESOURCES FOR EXPRESSION |
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Following the decade of comprehensive FL-cDNA collections, genome sciences entered into the phase of functional study, which utilizes FL-cDNA clones for various aims. To facilitate functional studies, cDNA inserts especially of ORFs (protein-coding regions) should be easily re-cloned in various vectors that allows researchers, for example, to express proteins in Escherichia coli and to express them as a GFP-fusion form in a mammalian cell. The MGC ORFeome project, an international activity, is establishing valuable resources of ORF entry clones for all human protein-coding genes (http://orfeomecollaboration.org/html/index.shtml).
Technologies for ORF entry clones
An ORF entry clone is generally defined as a cDNA clone from which an ORF region is easily transferred into various vectors. The technologies for exchanging vectors promise to promote the functional study of genes due to their ability for easy and high-throughput transfer of inserts. Several principles are available. The Gateway system that utilizes specific recombination reactions of lambda phage integration/excision at att sites is most widely used [19]. Notably, the Gateway system has been used for ORFeome projects to produce an ORF entry clone collection of all human protein-coding genes. The Cre-LoxP system is also available (www.clontech.com/clontech/products/families/creator/index.shtml). As an alternative, a simple endonuclease conversion system called the ‘super rare cutter (SRC) system’ was designed for ORF cloning, briefly outlined in Figure 1. Herein, two homing endonuclease recognition sites introduced at both flanking regions of the ORF allows its easy extraction and transfer to target vectors with a single reaction in one tube. Two DNA molecules of the amplified ORF fragment and the target vector plasmid can be simultaneously digested and ligated in one tube, since the ccdB gene avoids the appearance of background E. coli growth. Furthermore, homing endonucleases recognize very long nucleotide sequences. Thereby, unexpected digestion in target fragments of ORF does not occur. I-CeuI and PI-SceI recognize 26 bases and 39 bases, respectively. On the other hand, the big advantage of the Gateway system is the availability of various expression vectors, i.e. expression vectors for E. coli, baculo virus and mammalian cell.
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Establishment of ORF clones of Pyrococcus horikoshii
By using the SRC system, comprehensive ORF clones of P. horikoshii genes were established. Pyrococcus horikoshii shinkaj OT3 was isolated from a hydrothermal vent at a depth of 1395 m in the Okinawa Trough in the Pacific Ocean in 1992. It is a hyperthermophilic and anaerobic archaebacterium that grows optimally at 98°C in the presence of sulfur. The outstanding stability of the extreme-thermophile proteins helps the handling of the expressed proteins, especially in the purification step, followed by functional and structural genomic studies. Out of 2061 genes predicted from 1.74 Mb genome sequences [27], 1702 (83%) were cloned successfully in the SRC system. The clone insert size ranges from 150 bp to 3363 bp, with an average of 694 bp. After re-sequencing of the transferred ORF, expression of proteins in E. coli has been investigated. So far, 76 proteins have been expressed in soluble form, purified, crystallized and analysed for their structure. Furthermore, novel protein interactions were identified [22]. These results clearly show the usefulness of ORF clones.
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ESTABLISHMENT OF STANDARD CLONE BANKS AND ITS QUALITY CONTROL |
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For the distribution of clones, further steps are necessary: (i) the establishment of a standard bank, (ii) a continuous quality control and (iii) a feasible distribution system. Usually, established clones are stored at –80°C, randomly located in many multi-well titre plates. Often, unsuccessful or problematic clones are stored on the same plates. A standard set should be created by re-arraying non-redundant and successful clones into final plates. This step is essential to establish high quality and reliable clone banks as described subsequently.
Unexpected errors, such as the loss of viability of E. coli and mismatches between clones and sequence information, have often occurred in clone production and distribution. The most problematic step for clone distribution lays in picking up target clones from the huge number of frozen clones. A well-established non-redundant set of clones could minimize possible trouble. Furthermore, there should be a complete control for a standard clone bank to make ensure that it does not include any mismatch between clone and sequence information. In the case of the RIKEN FANTOM project, clones were sequenced twice. After the first sequencing they were submitted to a public DNA database. In the second step, the 103 000 full-sequenced cDNA clones were re-arrayed into the final multi-well titre plates for the standard set. Then they were re-sequenced both, from the 5'- and from the 3'-ends and by this the identities for the sequences deposited to public DNA database were controlled. When mismatches were found, we immediately go to the original plates tracking back through all the operations from re-arraying to re-sequencing, and choose the correct clone that corresponds to the sequence of the DNA database. Mismatches of clones and sequences observed in the distribution are very rare, about 1–3%. In contrast, higher error rates were observed for human FL-cDNA clones (9% of FLJ and 30% of MGC).
The DNA Book is one of the most reliable distribution methods to avoid potential problems with mismatches. In this book, thousands of plasmid DNAs are attached to pages of water soluble paper (Figure 2). The DNA Book users extract a plasmid of interest from the page by cutting out a plasmid-spotted area from a page and solving it in water. The DNA Book is easy to ship by courier for distributors and easy to store, since spotted plasmids in the DNA book are stable at room temperature [28, 29]. Certainly, a DNA Book containing a full set of cDNA clones established in the cDNA projects offers an ultimate clone resource for functional genomics. If a DNA Book is carefully made and checked not to contain errors, it avoids almost all potential problems that can occur during the clone distribution. Once the users have a DNA Book, they have all the clones in their hand and there is no need for ordering other clones. DNA Books containing FANTOM mouse FL-cDNA clones (ca 60 000 clones), rice FL-cDNA clones (ca 30 000 clones), Arabidopsis FL-cDNA clones (ca 1000 clones) and Pyrococcus ORF clones (ca 1700) have been released (http://genome.gsc.riken.jp/DNA-Book/).
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AVAILABILITY OF cDNA CLONE RESOURCES |
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The research community should have unrestrained access to cDNA clone resources, which is the infrastructure of functional genomics. It provides the experimental tools to facilitate an understanding of gene function. Large-scale projects for establishing resources have been conducted for this primary reason. In this sense, biological materials as well as the cDNA clones should be made available to the scientific community with minimal constraint [30, 31]. The MGC project led by Dr Gary Temple (http://orfeomecollaboration.org/html/index.shtml), is setting a good precedent by implementing a policy for a new clone resource to be available without any restrictions for both academic researchers and for-profit entities.
However, this liberal policy is not always possible due to issues that arise from intellectual property rights. There are commercial vendors of reagents who do not allow the free distribution without their permission of research products obtained by using their reagents [32]. An example is the expression vector pRETRO-SUPER, a product of Cancer Research Technology Limited (CRT), which is a very effective vector for expression of shRNA (short hair-pin RNA). Using this, the GNP has generated a comprehensive panel of shRNA expression vectors for about 20 000 human genes. Technology based on shRNA expression is a powerful tool to knock down the expression of specific genes in living cells. At the current time, the shRNA resources prepared by the GNP (Japan) can be shared only by the GNP members, and cannot be provided to labs outside the consortium due to an agreement negotiated with CRT. It is reasonable that commercial entities holding patents are entitled to get reasonable benefit for their intellectual property rights. However, it is also reasonable to expect that the activity of the scientific community should not be blocked or delayed, especially for key tool sets like clone resources which drive discovery and can provide great benefits to science and mankind.
Another issue is a matter of reach-through intellectual property that is burdening cDNA clones. In the case of rice FL-cDNA clones, NIAS insists on the retention of rights on patents generated in downstream research using their clones, even though the NIAS did not directly participate in the research. This seems to reduce the value of the resource, forcing many researchers to re-clone the cDNA to avoid any strings on the intellectual property rights. This is clearly wasteful and non-sensible. Biological resources should be provided to at least academia with minimal strings attached to promote discovery and to not waste resources. Persons in charge of planning and managing the projects aiming at establishing biological resources for research should understand these existing barriers in advance and be careful so that resources created can be made available freely to the community and promote scientific progress.
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MISSING cDNA CLONE RESOURCES |
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As introduced earlier, many cDNA clone resources have been established. Recently, however, the transcriptome especially of mammals has been revealed to be more complex than expected [33, 34]. Even in the RIKEN FANTOM project, only poly-adenylated mRNAs were collected, indicating that poly-A minus RNAs are still missing. In addition, many reports suggest the functional importance of small RNA shorter than 100 bp [35]. To understand biological phenomena at a molecular level, there is no doubt that continuous efforts are necessary to establish more comprehensive cDNA clone resources for functional genomics.
Key Points
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ACKNOWLEDGEMENTS |
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We thank all of the members of the RIKEN GSC-GERG and GSL and the FANTOM consortium members, K. Takio, C. Kuroishi and M. Miyano for data production, analysis and discussions, and C. Daub, K. Koseki, M. Persson, H. Daub and M. Nishikawa for their assistance in preparing the article and P. Cizdziel for critical suggestion. This work was supported by a Research Grant for National Project on Protein Structural and Functional Analysis from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government (MEXT) to Y.H., a Research Grant for the RIKEN Genome Exploration Research Project from MEXT to Y.H. and a grant of the Genome Network Project from MEXT to Y.H.
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FOOTNOTES |
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Jun Kawai is a Deputy Project Director at RIKEN Genomic Sciences Center, RIKEN, Japan. He earned his PhD from the University of Kyoto in 1991. His main area is genome science and mammalian transcriptome analysis.
Piero Carninci is Senior Scientist at RIKEN. After the Doctoral degree at University of Trieste, Italy (1989), he moved to Japan in 1995 with the mission to develop key technologies for genome and transcriptome analysis.
Yoshihide Hayashizaki is the Director at RIKEN Genomic Sciences Center, RIKEN, Japan. He received his MD and PhD from Osaka University Medical School in 1982 and 1986. He is organizing the FANTOM consortium. His main area is genome science.
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