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The twin pillars of GFAcT screening technology are the ability to perform cell-based assays in 96- or 384-well plate format and the availability of arrayed bioreagent collections to be screened for function which, for the Institute’s purposes, include gene-encoding cDNAs in promoter-bearing vectors (cDNAs), short-hairpin RNA-encoding cDNAs in promoter-bearing vectors (shRNAs), or synthetic small interfering RNAs (siRNAs). cDNAs can be obtained from several commercial sources or constructed de novo. shRNAs are to date only available by cloning them de novo. siRNAs are available from several vendors and collections of arrayed siRNAs targeting particular genes are soon to be available from QIAgen and others. For discussions herein, no attention will be paid to pooled libraries or other screening methodologies employing collections arrayed such that biogreagents in each well are heterogenous.
Procuring cDNA Clone Collections
Three principal collections are employed for GFAcT cDNA functional screening at this Institute, the Novartis Functional Genomics Area (NFGA) collection, the Mammalian Genome Collection (MGC), and the Origene TrueClone collection.
Novartis Functional Genomics
Area (NFGA) cDNA Collection
Description: The NFGA clone collection contains 20,704 individual clones in Gateway-adapted pCMVsport6 encoding approximately 14,000 unique human transcripts, >95% of which are full-length. Pre-annotation includes 5’ sequencing only. Number of arrayed unique transcripts: ~14,000 Organism: human Availability: Currently not publicly available. Contact information: Dr. Chuanzheng Song
Mammalian Genome Collection (MGC) cDNA
Collection
Description: As of August 2003, the MGC cDNA Collection contained >22,000 individual clones in five different vectors from two mammalian species, each clone encoding a fully sequenced unique transcript. Only one of the vectors contains a mammalian (CMV) promoter and is therefore suitable for the sorts of functional retrotransfection studies in mammalian cells described in our recent PNAS paper. Only these “transfectable” clones will be considered further. This vector, pCMVsport6, is Gateway adapted. The cDNA Cloning and Production group at GNF ( orth@gnf.org ) is currently transferring >8,000 human cDNAs from the promoterless pOTB7 vector to pCMVsport6, thus increasing total number of fully-sequenced human genes screenable to >12,000 unique transcripts. Number of arrayed unique transcripts: 3952 human, 7603 mouse Organism: human and mouse. Availability: Commercially available from three sources, American Type Tissue Culture, Open Biosystems, and Research Genetics/Invitrogen, although all clones in use at GNF have been purchased exclusively from American Type Tissue Culture (ATCC). ATCC sells clones both individually and in the same IRAK and IRAL 384-well format as MGC employs when full-length sequencing I.M.A.G.E. consortium clones (see http://image.llnl.gov/ and http://mgc.nci.nih.gov/ for detailed information on I.M.A.G.E. and MGC clones). In this format, full-length, fully sequence-validated human and mouse genes can be purchased for ~ $3 per gene, with the average plate containing ~230 full-length genes. Contact Information: ATCC (http://www.atcc.org
) Description: The Origene TrueClone Collection consists of
more than 20,000 unique human cDNA clones in three CMV promoter-driven non-Gateway
adapted vectors. Our Institute possesses and screens a subset of this collection
comprising the first 20,000 cDNAs. Short hairpin RNAs are typically encoded by cloned DNA oligos transcribed under the control of a RNA polymerase III promoter such as the human H1 or mouse U6 promoters. The DNA oligos are designed such that when transcribed they encode a 19mer with perfect homology to target gene sequences, a non-specific loop, and the 19mer’s complementary sequence, and a transcriptional termination signal. Upon transcription, the complementary halves of the single-stranded RNA transcript hybridize, forming a double-stranded 19mer joined by a single-stranded loop. Compelling reports of results obtained in knocking down target genes with specific shRNA constructs have recently been reported and any variety of shRNA cloning strategies can be employed. Given the plethora of alternatives in generating and/or cloning shRNAs and the likelihood of a general review appearing soon, no further details will be provided here. Some useful URLs detailing the many alternative strategies are:
http://sitemaker.umich.edu/dlturner.vectors/
files/u6_hairpin_sirna_6_21_02.doc
Unlike shRNAs, which are transcribed from DNA vectors designed
to encode a double-stranded >19mer product, siRNAs
are synthesized as two complementary RNA oligos
whose sequence is homologous and complementary to
the sense strand of the target gene and annealed
for use. This Institute’s collection of siRNAs were
designed using modified Tuschl rules (see
http://lion.gnf.org
for details) and synthesized largely by QIAgen
(
http://www1.qiagen.com/genexpression/gene_silencing/default.aspx
). Alternate vendors include IDT (
http://www.idtdna.com
) and Dharmacon (
http://www.dharmacon.com
). |
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2003 Genomics Institute of the Novartis Research Foundation. All Rights Reserved. |