easy to determine if an ESt is likely to contain a full-length sequence if it is derived from a directional oligo dT primed library and sequenced from the 5 end by searching for an ATG and an upstream stop codon. Once you identify a full-length EST, you should then be able to obtain the corresponding IMAGE clone from Incyte Genomics, Life Seq Public Incyte clones (http.//www.incyte.com/reagents/index.shtml),ResearchGenetics (http://www.resgen.com),ortheamEricanTypeCultureCollection (atcC,http://www.atcc.org).Ifthegeneispublishedyoucanalso try contacting the author who cloned it in order to obtain a cDNA clone. Most labs, including both academic and pharmaceutical/ biotech companies, will honor a request for a cDNA clone if it is published. Alternatively, you may consider deriving the gene de novo by rT-PCR using the sequence obtained above Depending on the size, abundance, and tissue distribution of the nRNA, a PCR approach could be straightforward or complex One may isolate RNA from tissue, generate cDNA from the RnA using reverse transcriptase, design PCR primers to perform PCR, and fish out the gene of interest. Alternatively, one may simply purchase a cDNA library from which to PCR amplify the gene Several vendors carry a wide array of high-quality cDNA libraries derived from human and animal tissues. For example, CDNA libraries for virtually every major human or murine tissue/organ canbeobtainedfromInvitrogen(http://www.invitrogen.com./ atalog_project/index.htmlorClontech(http://www.clontech.comm products/catalog/Libraries/index. html). These companies obtain heir samples from sources under Federal Guidelines. i Expression Vector Design and Subcloning Perhaps the most critical step in the process of expressing a gene is the vector design and subcloning. As much an art as a science, it nevertheless requires complete precision. In many cases you will need to amplify the gene by PCR from RNA. If the gene is in a library, you may also need to trim the 5 and 3 UTR (untranslated region) and to add restriction sites and/or a signal sequence if one is not already present. You may also want to add sEditor's note: In addition to the planning recommended by the authors, it is wise to ask commercial suppliers of expression systems about the existence of patents relating to the components of an expression vector(i.e, promoters) or the use of proteins produced by a patented expression vector/system Trill et aleasy to determine if an EST is likely to contain a full-length sequence if it is derived from a directional oligo dT primed library and sequenced from the 5¢ end by searching for an ATG and an upstream stop codon. Once you identify a full-length EST, you should then be able to obtain the corresponding IMAGE clone from Incyte Genomics, LifeSeq Public Incyte clones (http://www.incyte.com/reagents/index.shtml), Research Genetics (http://www.resgen.com), or the American Type Culture Collection (ATCC, http://www.atcc.org). If the gene is published, you can also try contacting the author who cloned it in order to obtain a cDNA clone. Most labs, including both academic and pharmaceutical/ biotech companies, will honor a request for a cDNA clone if it is published. Alternatively, you may consider deriving the gene de novo by RT-PCR using the sequence obtained above. Depending on the size, abundance, and tissue distribution of the mRNA, a PCR approach could be straightforward or complex. One may isolate RNA from tissue, generate cDNA from the RNA using reverse transcriptase, design PCR primers to perform PCR, and fish out the gene of interest. Alternatively, one may simply purchase a cDNA library from which to PCR amplify the gene. Several vendors carry a wide array of high-quality cDNA libraries derived from human and animal tissues. For example, cDNA libraries for virtually every major human or murine tissue/organ can be obtained from Invitrogen (http://www.invitrogen.com./ catalog_project/index.html) or Clontech (http://www.clontech.com/ products/catalog/Libraries/index.html). These companies obtain their samples from sources under Federal Guidelines.* Expression Vector Design and Subcloning Perhaps the most critical step in the process of expressing a gene is the vector design and subcloning. As much an art as a science, it nevertheless requires complete precision. In many cases you will need to amplify the gene by PCR from RNA. If the gene is in a library, you may also need to trim the 5¢ and 3¢ UTR (untranslated region) and to add restriction sites and/or a signal sequence if one is not already present. You may also want to add 498 Trill et al. *Editor’s note: In addition to the planning recommended by the authors, it is wise to ask commercial suppliers of expression systems about the existence of patents relating to the components of an expression vector (i.e., promoters) or the use of proteins produced by a patented expression vector/system