Some tips on making/purifying DNA for electroporation into ES cells: Some labs use the Cesium Chloride method for isolating DNA for electroporation however I find that this technique works in some peoples hands but does not work well in other peoples hands...everyone gets DNA but in some cases this DNA is "dirty" and results in very low transfection efficiencies. A Quiagen maxiprep followed by phenol/chloroform extraction, chloroform extraction and ethanol precipitation will give you DNA of sufficient quality for subsequent (after linearization) electroporation into ES cells. Other commercially available resin/column based plasmid DNA isolation kits are probably just as good for this purpose as the Quiagen kit. The final size of the targeting vector is often over 20-kb and I've found that to get a good yield from the Quiagen prep one needs to modify their P1 solution before use as follows: -add glucose (final amount in P1 solution 1% ie. 1 gram/100mls) -add lysozyme (final amount in P1 solution 5mg/ml) The 2 e-mails are as follows: 1st e-mail: October 1995 Gene targeters: A couple of years ago I compiled the sequences of the PGKneo and PGKtk cassettes that make up pPNT [ref: Tybulewicz et al. Cell vol 65 p11531163 (1991)]. This compilation contained quite a bit of unpublished sequence data and vector construction info that I got from a number of labs (the PGK promoter and terminator sequences that are in GenBank contain errors). At any rate, there were a number of requests for copies of it so I ended up e-mailing it out to a bunch of knock-out labs. For practical purposes this document is now kind of generic in form in that it contains the sequence of pPNT, the sequences of 2 additional pPNT based vectors: pPN2T [ref: Paszty et al. Nature Genetics vol 11 p33-39 (1995)] and pPN2T-hGHterm [ref: Zhu et al. Blood vol 93 p2404-2410 (1999)], as well as some general things to keep in mind when deciding on a targeting strategy and when building targeting vectors. Please pass this document on to any interested colleagues. Chris Paszty, Ph.D. Human Genome Center Lawrence Berkeley Laboratory Berkeley, CA 94720...U.S.A. Address as of February 1998 Amgen Inc. Dept. of Molecular Genetics One Amgen Center, MS 14-1-B Thousand Oaks, CA 91320-1789 U.S.A. tel: (805) 447-2082 fax: (805) 447-1982 e-mail: cpaszty@amgen.com Contributors of unpublished data/info: Garry Hannan, CSIRO, Australia: PGK-1 promoter and terminator sequences Victor Tybulewicz, MRC, England: details of construction of pPNT Karen Jardine and Michael McBurney, U. of Ottawa, Canada: details of construction of pKJ1 Some general info: pPNT is a pUC/Bluescript based vector...see Cell paper. The most effective way to use pPNT and pPNT-derivatives is to clone the short arm (1-2 kb) in-between the PGKneo cassette and the PGKtk cassette, and then to clone the long arm (6-11 kb) in-between the other side of the PGKneo cassette (PGK promoter side) and the unique Not I site. For electroporation the resulting targeting vector can then be linearized at the unique Not I site. In an effort to increase the effectiveness of positive-negative selection I've introduced a second same orientation PGKtk cassette into the single HindIII site of pPNT to create pPN2T. The apparent negative selection enrichment obtained with an alpha globin targeting vector (pPN2T-alphaKO ) based on pPN2T was 3-5 fold higher than that obtained with a variety of different single PGKtk based targeting vectors that had been electroporated and selected under identical conditions as those used for pPN2T-alphaKO ie. same lab, same E.S. cells, same feeder cells, same reagents and same protocols. Although this has not been rigorously tested it is likely that the additional PGKtk cassette present in pPN2T-alphaKO is responsible for the high apparent enrichment values obtained with this targeting vector. Creating a null allele knock-out for small genes is usually not too difficult as one can just delete the whole gene or most of it to guarantee achieving complete loss of gene function. However, for larger genes, particularly those who's genomic structure has not been well mapped out, creating this type of whole gene deletion can be time consuming and difficult. The targeting strategy we've been using for such large genes is to create as extensive a deletion as is practical in the 5' region of the gene. The deletion is designed so as to include the presumed translational initiator codon and at least 1 kb of sequence upstream from the ATG in an effort to delete the promoter and thereby shut down transcription. Permanently shutting down transcription is an extremely effective way to achieve complete loss of gene function. In situations where transcription isn't shut down and a full length but mutated mRNA is produced one runs the risk that the mutagenic (translational stop codons in 3 frames etc...) neo cassette might be removed by normal or alternative splicing pathways. This may result in a "leaky" mutation rather than a null mutation or, even worse, a dominant negative mutation. Because some genes have multiple promoters which may be quite a distance upstream of the ATG, deleting a 1 kb region upstream of the ATG, even if it does inactivate one of the promoters, might not cause a complete shut down of transcription. An additional "tool" for shutting down transcription is transcriptional termination. For this reason it is desirable to end up with a targeting event that results in the insertion of a polyA-signal/terminator in the same orientation as the transcription of the gene in question. For pPNT and pPN2T if one's long arm is 5' of one's short arm within the gene to be inactivated then the structure of the final targeting event puts the PGKneo cassette in the same transcriptional orientation as that of the gene's. In this case, transcripts originating from the gene's promoter(s) will terminate at the PGK polyA-signal/terminator that is part of the PGKneo cassette. This leaves any downstream exons untranscribed and thus increases the probability of creating a null allele. However, for pPNT and pPN2T, if one's short arm is 5' of one's long arm within the gene to be inactivated then the structure of the final targeting event will put the PGKneo cassette in reverse transcriptional orientation with respect to that of the gene's. Thus the extra protection against the production of a "leaky" allele that is afforded by the premature termination of any transcripts originating from the gene's promoter(s) is lost. To retain the feature of transcriptional termination for this configuration of long and short arms we've introduced the polyA-signal/terminator from the human growth hormone N gene into pPN2T to create pPN2T-hGHterm. Although this is a human and not a murine polyA-signal/terminator, it is highly homologous to other mammalian growth hormone polyA-signal/terminators, has been extensively used in adenovirus based gene therapy vectors in mice and thus should work well for terminating transcription at a targeted locus. The standard null allele type knock-out is usually the most straight forward to generate, however other approaches that should be considered are: 1) The inclusion of a reporter gene, such as lacZ (for example: Le Mouellic et al 1992 Cell vol 69, p246-251; Mountford et al 1994 PNAS vol 91 p4303-4307) in one's targeting vector. This allows one to examine the spatial and temporal pattern of gene expression of the targeted gene and also to follow cell fate. 2) A conditional knock-out using Cre-loxP (Gu et al 1994 Science vol 265 p103-106; Kuhn et al 1995 Science vol 269, p1427-1429) and/or FLP-frt (Fiering et al 1995 Genes and Development vol 9, p2203-2213). Cell fate can also be followed using these recombinase systems. Other variations on the basic knock-out theme will undoubtedly be developed in the future, so it's wise to check the latest literature. The particular approach one decides on is based on what information one is interested in, as well as how much of a hurry one is in. The standard null allele type is still the simplest and quickest to make. For many genes a fair amount of information can be gotten from a null allele knock-out. If one is still intrigued then a subsequent fancier knock-out can be generated. The sequences and reference points for pPNT, pPN2T and pPN2T-hGHterm are as follows: Reference points for the pPNT sequence (bprs): 7-23 T3 promoter in pBluescript SK(+) 54-61 single NotI site in pPNT 71-78 single Sse8387I site in pPNT 81-587 EcoRI(destroyed)-TaqI fragment of PGK-1 promoter 589-1418 PstI-HincII(destroyed) neo gene containing frag from pKJ1DB (derived from pKJ1 which was derived from pMC1neo). 600-602 neo start codon 1401-1403 neo stop codon 1419-1883 PvuII(destroyed)-HindIII(filled) frag of PGK-1 terminator 1914-1919 single EcoRI site in pPNT 1914-2421 EcoRI-TaqI fragment of PGK-1 promoter 2423-4253 PstI-PvuII(destroyed) frag from HSV-1 containing the tk gene. 2519-2521 tk start codon 3647-3649 tk stop codon 4257-4714 PstI-HindIII fragment of PGK-1 terminator 4714-7348 pUC18/Bluescript SK+ vector backbone pPNT sequence: GCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGT GGCGGCCGCTCGAGGGCCCCTGCAGGTCAATTCTACCGGGTAGGGGAGGCGC TTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGCACTTGGC GCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGCGC CAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTA GTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAA TGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCA ATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTC GCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGG GCGGGCTCAGGGGCGGGGCGGGCGCGAAGGTCCTCCCGAGGCCCGGCATTCT CGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCC GGGCCTTTCGACCTGCAGCCAATATGGGATCGGCCATTGAACAAGATGGATT GCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGG GCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGC AGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAA CTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTT GCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATT GGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAG AAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGG CTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTAC TCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAG GGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACG GCGATGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTG GAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGG ACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGG CGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATT CGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGGGGATCGA TCCGTCCTGTAAGTCTGCAGAAATTGATGATCTATTAAACAATAAAGATGTCC ACTAAAATGGAAGTTTTTCCTGTCATACTTTGTTAAGAAGGGTGAGAACAGA GTACCTACATTTTGAATGGAAGGATTGGAGCTACGGGGGTGGGGGTGGGGTG GGATTAGATAAATGCCTGCTCTTTACTGAAGGCTCTTTACTATTGCTTTATGA TAATGTTTCATAGTTGGATATCATAATTTAAACAAGCAAAACCAAATTAAGG GCCAGCTCATTCCTCCCACTCATGATCTATAGATCTATAGATCTCTCGTGGGA TCATTGTTTTTCTCTTGATTCCCACTTTGTGGTTCTAAGTACTGTGGTTTCCA AATGTGTCAGTTTCATAGCCTGAAGAACGAGATCAGCAGCCTCTGTTCCAC ATACACTTCATTCTCAGTATTGTTTTGCCAAGTTCTAATTCCATCAGAAGCTG ACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTC TACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAG CAGCCCCGCTGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACA TTCCACATCCACCGGTAGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGC GCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCG CGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCA GATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGG CCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTG GGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCGAAGG TCCTCCCGAGGCCCGGCATTCTCGCACGCTTCAAAAGCGCACGTCTGCCGCGC TGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCGACCCGCTTAACA GCGTCAACAGCGTGCCGCAGATCTTGGTGGCGTGAAACTCCCGCACCTCTTC GGCCAGCGCCTTGTAGAAGCGCGTATGGCTTCGTACCCCTGCCATCAACACG CGTCTGCGTTCGACCAGGCTGCGCGTTCTCGCGGCCATAACAACCGACGTAC GGCGTTGCGCCCTCGCCGGCAACAAAAAGCCACGGAAGTCCGCCTGGAGCAG AAAATGCCCACGCTACTGCGGGTTTATATAGACGGTCCCCACGGGATGGGGA AAACCACCACCACGCAACTGCTGGTGGCCCTGGGTTCGCGCGACGATATCGT CTACGTACCCGAGCCGATGACTTACTGGCGGGTGTTGGGGGCTTCCGAGACA ATCGCGAACATCTACACCACACAACACCGCCTCGACCAGGGTGAGATATCGG CCGGGGACGCGGCGGTGGTAATGACAAGCGCCCAGATAACAATGGGCATGC CTTATGCCGTGACCGACGCCGTTCTGGCTCCTCATATCGGGGGGGAGGCTGG GAGCTCACATGCCCCGCCCCCGGCCCTCACCCTCATCTTCGACCGCCATCCCA TCGCCGCCCTCCTGTGCTACCCGGCCGCGCGATACCTTATGGGCAGCATGACC CCCCAGGCCGTGCTGGCGTTCGTGGCCCTCATCCCGCCGACCTTGCCCGGCAC AAACATCGTGTTGGGGGCCCTTCCGGAGGACAGACACATCGACCGCCTGGCC AAACGCCAGCGCCCCGGCGAGCGGCTTGACCTGGCTATGCTGGCCGCGATTC GCCGCGTTTATGGGCTGCTTGCCAATACGGTGCGGTATCTGCAGGGCGGCGG GTCGTGGCGGGAGGATTGGGGACAGCTTTCGGGGGCGGCCGTGCCGCCCCAG GGTGCCGAGCCCCAGAGCAACGCGGGCCCACGACCCCATATCGGGGACACGT TATTTACCCTGTTTCGGGCCCCCGAGTTGCTGGCCCCCAACGGCGACCTGTAT AACGTGTTTGCCTGGGCTTTGGACGTCTTGGCCAAACGCCTCCGTCCCATGCA TGTCTTTATCCTGGATTACGACCAATCGCCCGCCGGCTGCCGGGACGCCCTGC TGCAACTTACCTCCGGGATGGTCCAGACCCACGTCACCACCCCAGGCTCCAT ACCGACGATCTGCGACCTGGCGCGCACGTTTGCCCGGGAGATGGGGGAGGCT AACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGC AATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACG CGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCAT TGGGACCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCAACCCCCAAGTTC GGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAAGCCCTGCCAT AGCCACGGGCCCCGTGGGTTAGGGACGGGGTCCCCCATGGGGAATGGTTTAT GGTTCGTGGGGGTTATTATTTTGGGCGTTGCGTGGGGTCAGGTCCACGACTGG ACTGAGCAGACAGACCCATGGTTTTTGGATGGCCTGGGCATGGACCGCATGT ACTGGCGCGACACGAACACCGGGCGTCTGTGGCTGCCAAACACCCCCGACCC CCAAAAACCACCGCGCGGATTTCTGGCGCCGCCGGACGAACTAAACCTGACT ACGGCATCTCTGCCCCTTCTTCGCTGGTACGAGGAGCGCTTTTGTTTTGTATTG GTCACCACGGCCGAGTTTCCGCGGGACCCCGGCCAGGACCTGCAGAAATTGA TGATCTATTAAACAATAAAGATGTCCACTAAAATGGAAGTTTTTCCTGTCATA CTTTGTTAAGAAGGGTGAGAACAGAGTACCTACATTTTGAATGGAAGGATTG GAGCTACGGGGGTGGGGGTGGGGTGGGATTAGATAAATGCCTGCTCTTTACT GAAGGCTCTTTACTATTGCTTTATGATAATGTTTCATAGTTGGATATCATAATT TAAACAAGCAAAACCAAATTAAGGGCCAGCTCATTCCTCCCACTCATGATCT ATAGATCTATAGATCTCTCGTGGGATCATTGTTTTTCTCTTGATTCCCACTTTG TGGTTCTAAGTACTGTGGTTTCCAAATGTGTCAGTTTCATAGCCTGAAGAACG AGATCAGCAGCCTCTGTTCCACATACACTTCATTCTCAGTATTGTTTTGCCAA GTTCTAATTCCATCAG AAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGA AAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCG CCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAG TTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTAC GCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCT GCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCT GACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGC TGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC CGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTT AATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGG AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATA TGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA AAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTT TTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGA AAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAA CTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACG TTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTAT CCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCT CAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGA TGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATA ACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTA ACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTG GGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTA CTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAA AGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTG CTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATA CTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAA GATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGT TCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGAT CCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCT ACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGA AGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTG TAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATA CCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGT CGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAG CGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAAC GACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCA CGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTC GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCT TTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGT GATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCC TTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCC TGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAG CTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGC GAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTG GCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGG GCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCC CAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAG CGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAA Reference points for the pPN2T sequence (bprs): 7-23 T3 promoter in pBluescript SK(+) 54-61 single NotI site in pPN2T 71-78 single Sse8387I site in pPN2T 81-587 EcoRI(destroyed)-TaqI fragment of PGK-1 promoter 589-1418 PstI-HincII(destroyed) neo gene containing frag from pKJ1DB (derived from pKJ1 which was derived from pMC1neo). 600-602 neo start codon 1401-1403 neo stop codon 1419-1883 PvuII(destroyed)-HindIII(filled) frag of PGK-1 terminator 1914-1919 single EcoRI site in pPN2T 1914-2421 EcoRI-TaqI fragment of PGK-1 promoter 2423-4253 PstI-PvuII(destroyed) frag from HSV-1 containing the tk gene. 2519-2521 tk start codon 3647-3649 tk stop codon 4257-4714 PstI-HindIII(filled) fragment of PGK-1 terminator 4718-5225 EcoRI(filled)-TaqI fragment of PGK-1 promoter 5227-7057 PstI-PvuII(destroyed) frag from HSV-1 containing the tk gene. 5323-5325 tk start codon 6451-6453 tk stop codon 7061-7518 PstI-HindIII(filled) fragment of PGK-1 terminator 7518-10152 pUC18/Bluescript SK+ vector backbone pPN2T sequence: GCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGT GGCGGCCGCTCGAGGGCCCCTGCAGGTCAATTCTACCGGGTAGGGGAGGCGC TTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGCACTTGGC GCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGCGC CAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTA GTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAA TGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCA ATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTC GCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGG GCGGGCTCAGGGGCGGGGCGGGCGCGAAGGTCCTCCCGAGGCCCGGCATTCT CGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCC GGGCCTTTCGACCTGCAGCCAATATGGGATCGGCCATTGAACAAGATGGATT GCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGG GCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGC AGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAA CTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTT GCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATT GGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAG AAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGG CTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTAC TCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAG GGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACG GCGATGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTG GAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGG ACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGG CGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATT CGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGGGGATCGA TCCGTCCTGTAAGTCTGCAGAAATTGATGATCTATTAAACAATAAAGATGTCC ACTAAAATGGAAGTTTTTCCTGTCATACTTTGTTAAGAAGGGTGAGAACAGA GTACCTACATTTTGAATGGAAGGATTGGAGCTACGGGGGTGGGGGTGGGGTG GGATTAGATAAATGCCTGCTCTTTACTGAAGGCTCTTTACTATTGCTTTATGA TAATGTTTCATAGTTGGATATCATAATTTAAACAAGCAAAACCAAATTAAGG GCCAGCTCATTCCTCCCACTCATGATCTATAGATCTATAGATCTCTCGTGGGA TCATTGTTTTTCTCTTGATTCCCACTTTGTGGTTCTAAGTACTGTGGTTTCCA AATGTGTCAGTTTCATAGCCTGAAGAACGAGATCAGCAGCCTCTGTTCCAC ATACACTTCATTCTCAGTATTGTTTTGCCAAGTTCTAATTCCATCAGAAGCTG ACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTC TACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAG CAGCCCCGCTGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACA TTCCACATCCACCGGTAGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGC GCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCG CGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCA GATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGG CCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTG GGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCGAAGG TCCTCCCGAGGCCCGGCATTCTCGCACGCTTCAAAAGCGCACGTCTGCCGCGC TGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCGACCCGCTTAACA GCGTCAACAGCGTGCCGCAGATCTTGGTGGCGTGAAACTCCCGCACCTCTTC GGCCAGCGCCTTGTAGAAGCGCGTATGGCTTCGTACCCCTGCCATCAACACG CGTCTGCGTTCGACCAGGCTGCGCGTTCTCGCGGCCATAACAACCGACGTAC GGCGTTGCGCCCTCGCCGGCAACAAAAAGCCACGGAAGTCCGCCTGGAGCAG AAAATGCCCACGCTACTGCGGGTTTATATAGACGGTCCCCACGGGATGGGGA AAACCACCACCACGCAACTGCTGGTGGCCCTGGGTTCGCGCGACGATATCGT CTACGTACCCGAGCCGATGACTTACTGGCGGGTGTTGGGGGCTTCCGAGACA ATCGCGAACATCTACACCACACAACACCGCCTCGACCAGGGTGAGATATCGG CCGGGGACGCGGCGGTGGTAATGACAAGCGCCCAGATAACAATGGGCATGC CTTATGCCGTGACCGACGCCGTTCTGGCTCCTCATATCGGGGGGGAGGCTGG GAGCTCACATGCCCCGCCCCCGGCCCTCACCCTCATCTTCGACCGCCATCCCA TCGCCGCCCTCCTGTGCTACCCGGCCGCGCGATACCTTATGGGCAGCATGACC CCCCAGGCCGTGCTGGCGTTCGTGGCCCTCATCCCGCCGACCTTGCCCGGCAC AAACATCGTGTTGGGGGCCCTTCCGGAGGACAGACACATCGACCGCCTGGCC AAACGCCAGCGCCCCGGCGAGCGGCTTGACCTGGCTATGCTGGCCGCGATTC GCCGCGTTTATGGGCTGCTTGCCAATACGGTGCGGTATCTGCAGGGCGGCGG GTCGTGGCGGGAGGATTGGGGACAGCTTTCGGGGGCGGCCGTGCCGCCCCAG GGTGCCGAGCCCCAGAGCAACGCGGGCCCACGACCCCATATCGGGGACACGT TATTTACCCTGTTTCGGGCCCCCGAGTTGCTGGCCCCCAACGGCGACCTGTAT AACGTGTTTGCCTGGGCTTTGGACGTCTTGGCCAAACGCCTCCGTCCCATGCA TGTCTTTATCCTGGATTACGACCAATCGCCCGCCGGCTGCCGGGACGCCCTGC TGCAACTTACCTCCGGGATGGTCCAGACCCACGTCACCACCCCAGGCTCCAT ACCGACGATCTGCGACCTGGCGCGCACGTTTGCCCGGGAGATGGGGGAGGCT AACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGC AATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACG CGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCAT TGGGACCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCAACCCCCAAGTTC GGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAAGCCCTGCCAT AGCCACGGGCCCCGTGGGTTAGGGACGGGGTCCCCCATGGGGAATGGTTTAT GGTTCGTGGGGGTTATTATTTTGGGCGTTGCGTGGGGTCAGGTCCACGACTGG ACTGAGCAGACAGACCCATGGTTTTTGGATGGCCTGGGCATGGACCGCATGT ACTGGCGCGACACGAACACCGGGCGTCTGTGGCTGCCAAACACCCCCGACCC CCAAAAACCACCGCGCGGATTTCTGGCGCCGCCGGACGAACTAAACCTGACT ACGGCATCTCTGCCCCTTCTTCGCTGGTACGAGGAGCGCTTTTGTTTTGTATTG GTCACCACGGCCGAGTTTCCGCGGGACCCCGGCCAGGACCTGCAGAAATTGA TGATCTATTAAACAATAAAGATGTCCACTAAAATGGAAGTTTTTCCTGTCATA CTTTGTTAAGAAGGGTGAGAACAGAGTACCTACATTTTGAATGGAAGGATTG GAGCTACGGGGGTGGGGGTGGGGTGGGATTAGATAAATGCCTGCTCTTTACT GAAGGCTCTTTACTATTGCTTTATGATAATGTTTCATAGTTGGATATCATAATT TAAACAAGCAAAACCAAATTAAGGGCCAGCTCATTCCTCCCACTCATGATCT ATAGATCTATAGATCTCTCGTGGGATCATTGTTTTTCTCTTGATTCCCACTTTG TGGTTCTAAGTACTGTGGTTTCCAAATGTGTCAGTTTCATAGCCTGAAGAACG AGATCAGCAGCCTCTGTTCCACATACACTTCATTCTCAGTATTGTTTTGCCAA GTTCTAATTCCATCAGAAGCTAATTC TACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAG CAGCCCCGCTGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACA TTCCACATCCACCGGTAGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGC GCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCG CGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCA GATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGG CCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTG GGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCGAAGG TCCTCCCGAGGCCCGGCATTCTCGCACGCTTCAAAAGCGCACGTCTGCCGCGC TGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCGACCCGCTTAACA GCGTCAACAGCGTGCCGCAGATCTTGGTGGCGTGAAACTCCCGCACCTCTTC GGCCAGCGCCTTGTAGAAGCGCGTATGGCTTCGTACCCCTGCCATCAACACG CGTCTGCGTTCGACCAGGCTGCGCGTTCTCGCGGCCATAACAACCGACGTAC GGCGTTGCGCCCTCGCCGGCAACAAAAAGCCACGGAAGTCCGCCTGGAGCAG AAAATGCCCACGCTACTGCGGGTTTATATAGACGGTCCCCACGGGATGGGGA AAACCACCACCACGCAACTGCTGGTGGCCCTGGGTTCGCGCGACGATATCGT CTACGTACCCGAGCCGATGACTTACTGGCGGGTGTTGGGGGCTTCCGAGACA ATCGCGAACATCTACACCACACAACACCGCCTCGACCAGGGTGAGATATCGG CCGGGGACGCGGCGGTGGTAATGACAAGCGCCCAGATAACAATGGGCATGC CTTATGCCGTGACCGACGCCGTTCTGGCTCCTCATATCGGGGGGGAGGCTGG GAGCTCACATGCCCCGCCCCCGGCCCTCACCCTCATCTTCGACCGCCATCCCA TCGCCGCCCTCCTGTGCTACCCGGCCGCGCGATACCTTATGGGCAGCATGACC CCCCAGGCCGTGCTGGCGTTCGTGGCCCTCATCCCGCCGACCTTGCCCGGCAC AAACATCGTGTTGGGGGCCCTTCCGGAGGACAGACACATCGACCGCCTGGCC AAACGCCAGCGCCCCGGCGAGCGGCTTGACCTGGCTATGCTGGCCGCGATTC GCCGCGTTTATGGGCTGCTTGCCAATACGGTGCGGTATCTGCAGGGCGGCGG GTCGTGGCGGGAGGATTGGGGACAGCTTTCGGGGGCGGCCGTGCCGCCCCAG GGTGCCGAGCCCCAGAGCAACGCGGGCCCACGACCCCATATCGGGGACACGT TATTTACCCTGTTTCGGGCCCCCGAGTTGCTGGCCCCCAACGGCGACCTGTAT AACGTGTTTGCCTGGGCTTTGGACGTCTTGGCCAAACGCCTCCGTCCCATGCA TGTCTTTATCCTGGATTACGACCAATCGCCCGCCGGCTGCCGGGACGCCCTGC TGCAACTTACCTCCGGGATGGTCCAGACCCACGTCACCACCCCAGGCTCCAT ACCGACGATCTGCGACCTGGCGCGCACGTTTGCCCGGGAGATGGGGGAGGCT AACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGC AATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACG CGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCAT TGGGACCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCAACCCCCAAGTTC GGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAAGCCCTGCCAT AGCCACGGGCCCCGTGGGTTAGGGACGGGGTCCCCCATGGGGAATGGTTTAT GGTTCGTGGGGGTTATTATTTTGGGCGTTGCGTGGGGTCAGGTCCACGACTGG ACTGAGCAGACAGACCCATGGTTTTTGGATGGCCTGGGCATGGACCGCATGT ACTGGCGCGACACGAACACCGGGCGTCTGTGGCTGCCAAACACCCCCGACCC CCAAAAACCACCGCGCGGATTTCTGGCGCCGCCGGACGAACTAAACCTGACT ACGGCATCTCTGCCCCTTCTTCGCTGGTACGAGGAGCGCTTTTGTTTTGTATTG GTCACCACGGCCGAGTTTCCGCGGGACCCCGGCCAGGACCTGCAGAAATTGA TGATCTATTAAACAATAAAGATGTCCACTAAAATGGAAGTTTTTCCTGTCATA CTTTGTTAAGAAGGGTGAGAACAGAGTACCTACATTTTGAATGGAAGGATTG GAGCTACGGGGGTGGGGGTGGGGTGGGATTAGATAAATGCCTGCTCTTTACT GAAGGCTCTTTACTATTGCTTTATGATAATGTTTCATAGTTGGATATCATAATT TAAACAAGCAAAACCAAATTAAGGGCCAGCTCATTCCTCCCACTCATGATCT ATAGATCTATAGATCTCTCGTGGGATCATTGTTTTTCTCTTGATTCCCACTTTG TGGTTCTAAGTACTGTGGTTTCCAAATGTGTCAGTTTCATAGCCTGAAGAACG AGATCAGCAGCCTCTGTTCCACATACACTTCATTCTCAGTATTGTTTTGCCAA GTTCTAATTCCATCAGAAGCTAGCTT GGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGA AAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCG CCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAG TTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTAC GCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCT GCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCT GACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGC TGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC CGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTT AATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGG AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATA TGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA AAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTT TTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGA AAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAA CTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACG TTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTAT CCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCT CAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGA TGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATA ACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTA ACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTG GGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTA CTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAA AGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTG CTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATA CTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAA GATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGT TCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGAT CCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCT ACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGA AGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTG TAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATA CCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGT CGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAG CGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAAC GACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCA CGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTC GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCT TTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGT GATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCC TTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCC TGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAG CTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGC GAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTG GCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGG GCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCC CAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAG CGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAA Reference points for the pPN2T-hGHterm sequence (bprs): 7-23 T3 promoter in pBluescript SK(+) 54-61 single NotI site in pPN2T-hGHterm 71-78 single Sse8387I site in pPN2T-hGHterm 81-587 EcoRI(destroyed)-TaqI fragment of PGK-1 promoter 589-1418 PstI-HincII(destroyed) neo gene containing frag from pKJ1DB (derived from pKJ1 which was derived from pMC1neo). 600-602 neo start codon 1401-1403 neo stop codon 1419-1883 PvuII(destroyed)-HindIII(filled) frag of PGK-1 terminator 1887-1892 single XbaI site in pPN2T-hGHterm 1893-2477 reverse orientation hGH-N gene polyA signal/terminator 2478-2483 single BamHI site in pPN2T-hGHterm 2498-2503 single EcoRI site in pPN2T-hGHterm 2498-3005 EcoRI-TaqI fragment of PGK-1 promoter 3007-4837 PstI-PvuII(destroyed) frag from HSV-1 containing the tk gene. 3103-3105 tk start codon 4231-4233 tk stop codon 4841-5298 PstI-HindIII(filled) fragment of PGK-1 terminator 5302-5809 EcoRI(filled)-TaqI fragment of PGK-1 promoter 5811-7641 PstI-PvuII(destroyed) frag from HSV-1 containing the tk gene. 5907-5909 tk start codon 7035-7037 tk stop codon 7645-8102 PstI-HindIII(filled) fragment of PGK-1 terminator 8102-10736 pUC18/Bluescript SK+ vector backbone pPN2T-hGHterm sequence: GCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGT GGCGGCCGCTCGAGGGCCCCTGCAGGTCAATTCTACCGGGTAGGGGAGGCGC TTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGCACTTGGC GCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGCGC CAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTA GTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAA TGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCA ATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTC GCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGG GCGGGCTCAGGGGCGGGGCGGGCGCGAAGGTCCTCCCGAGGCCCGGCATTCT CGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCC GGGCCTTTCGACCTGCAGCCAATATGGGATCGGCCATTGAACAAGATGGATT GCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGG GCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGC AGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAA CTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTT GCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATT GGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAG AAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGG CTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTAC TCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAG GGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACG GCGATGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTG GAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGG ACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGG CGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATT CGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGGGGATCGA TCCGTCCTGTAAGTCTGCAGAAATTGATGATCTATTAAACAATAAAGATGTCC ACTAAAATGGAAGTTTTTCCTGTCATACTTTGTTAAGAAGGGTGAGAACAGA GTACCTACATTTTGAATGGAAGGATTGGAGCTACGGGGGTGGGGGTGGGGTG GGATTAGATAAATGCCTGCTCTTTACTGAAGGCTCTTTACTATTGCTTTATGA TAATGTTTCATAGTTGGATATCATAATTTAAACAAGCAAAACCAAATTAAGG GCCAGCTCATTCCTCCCACTCATGATCTATAGATCTATAGATCTCTCGTGGGA TCATTGTTTTTCTCTTGATTCCCACTTTGTGGTTCTAAGTACTGTGGTTTCCA AATGTGTCAGTTTCATAGCCTGAAGAACGAGATCAGCAGCCTCTGTTCCAC ATACACTTCATTCTCAGTATTGTTTTGCCAAGTTCTAATTCCATCAGAAGCTG ACTCTAGA CAGGCATCTACTGAGTGGACCCAACGCATGAGAGGACAGTGCCAAGCAAGC AACTCAAATGTCCCACCGGTTGGGCATGGCAGGTAGCCTATGCTGTGTCTGG ACGTCCTCCTGCTGGTATAGTTATTTTAAAATCAGAAGGACAGGGAAGGGA GCAGTGGTTCACGCCTGTAATCCCAGCAATTTGGGAGGCCAAGGTGGGTAGA TCACCTGAGATTAGGAGTTGGAGACCAGCCTGGCCAATATGGTGAAACCCCG TCTCTACCAAAAAAACAAAAATTAGCTGAGCCTGGTCATGCATGCCTGGAAT CCCAACAACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCAGGAGGCG GAGATTGCAGTGAGCCAAGATTGTGCCACTGCACTCCAGCTTGGTTCCCGAA TAGACCCCGCAGGCCCTACAGGTTGTCTTCCCAACTTGCCCCTTGCTCCATAC CACCCCCCTCCACCCCATAATATTATAGAAGGACACCTAGTCAGACAAAATG ATGCAACTTAATTTTATTAGGACAAGGCTGGTGGGCACTGGAGTGGCAACTT CCAAGGCCAGGAGA GGATCCCCGGGTACCGAGCTCGAATTCTACCGGGTAGGGGAGGCGCTTTTCCCAA GGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGCACTTGGCGCTACACAAGT GGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGCGCCAACCGGCTCCGTTC TTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCC CGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACT AGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGG GGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAA GGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGC GAAGGTCCTCCCGAGGCCCGGCATTCTCGCACGCTTCAAAAGCGCACGTCTGCCG CGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCGACCCGCTTAACA GCGTCAACAGCGTGCCGCAGATCTTGGTGGCGTGAAACTCCCGCACCTCTTCGGC CAGCGCCTTGTAGAAGCGCGTATGGCTTCGTACCCCTGCCATCAACACGCGTCTGC GTTCGACCAGGCTGCGCGTTCTCGCGGCCATAACAACCGACGTACGGCGTTGCGC CCTCGCCGGCAACAAAAAGCCACGGAAGTCCGCCTGGAGCAGAAAATGCCCACGC TACTGCGGGTTTATATAGACGGTCCCCACGGGATGGGGAAAACCACCACCACGCA ACTGCTGGTGGCCCTGGGTTCGCGCGACGATATCGTCTACGTACCCGAGCCGATG ACTTACTGGCGGGTGTTGGGGGCTTCCGAGACAATCGCGAACATCTACACCACAC AACACCGCCTCGACCAGGGTGAGATATCGGCCGGGGACGCGGCGGTGGTAATGA CAAGCGCCCAGATAACAATGGGCATGCCTTATGCCGTGACCGACGCCGTTCTGGC TCCTCATATCGGGGGGGAGGCTGGGAGCTCACATGCCCCGCCCCCGGCCCTCACC CTCATCTTCGACCGCCATCCCATCGCCGCCCTCCTGTGCTACCCGGCCGCGCGATA CCTTATGGGCAGCATGACCCCCCAGGCCGTGCTGGCGTTCGTGGCCCTCATCCCG CCGACCTTGCCCGGCACAAACATCGTGTTGGGGGCCCTTCCGGAGGACAGACACA TCGACCGCCTGGCCAAACGCCAGCGCCCCGGCGAGCGGCTTGACCTGGCTATGCT GGCCGCGATTCGCCGCGTTTATGGGCTGCTTGCCAATACGGTGCGGTATCTGCAG GGCGGCGGGTCGTGGCGGGAGGATTGGGGACAGCTTTCGGGGGCGGCCGTGCC GCCCCAGGGTGCCGAGCCCCAGAGCAACGCGGGCCCACGACCCCATATCGGGGA CACGTTATTTACCCTGTTTCGGGCCCCCGAGTTGCTGGCCCCCAACGGCGACCTGT ATAACGTGTTTGCCTGGGCTTTGGACGTCTTGGCCAAACGCCTCCGTCCCATGCAT GTCTTTATCCTGGATTACGACCAATCGCCCGCCGGCTGCCGGGACGCCCTGCTGC AACTTACCTCCGGGATGGTCCAGACCCACGTCACCACCCCAGGCTCCATACCGAC GATCTGCGACCTGGCGCGCACGTTTGCCCGGGAGATGGGGGAGGCTAACTGAAA CACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGAC AGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCC AGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGACCAATACGCCCG CGTTTCTTCCTTTTCCCCACCCCAACCCCCAAGTTCGGGTGAAGGCCCAGGGCTCG CAGCCAACGTCGGGGCGGCAAGCCCTGCCATAGCCACGGGCCCCGTGGGTTAGG GACGGGGTCCCCCATGGGGAATGGTTTATGGTTCGTGGGGGTTATTATTTTGGGC GTTGCGTGGGGTCAGGTCCACGACTGGACTGAGCAGACAGACCCATGGTTTTTGG ATGGCCTGGGCATGGACCGCATGTACTGGCGCGACACGAACACCGGGCGTCTGT GGCTGCCAAACACCCCCGACCCCCAAAAACCACCGCGCGGATTTCTGGCGCCGCC GGACGAACTAAACCTGACTACGGCATCTCTGCCCCTTCTTCGCTGGTACGAGGAGC GCTTTTGTTTTGTATTGGTCACCACGGCCGAGTTTCCGCGGGACCCCGGCCAGGAC CTGCAGAAATTGATGATCTATTAAACAATAAAGATGTCCACTAAAATGGAAGTTTTT CCTGTCATACTTTGTTAAGAAGGGTGAGAACAGAGTACCTACATTTTGAATGGAAG GATTGGAGCTACGGGGGTGGGGGTGGGGTGGGATTAGATAAATGCCTGCTCTTTA CTGAAGGCTCTTTACTATTGCTTTATGATAATGTTTCATAGTTGGATATCATAATTTA AACAAGCAAAACCAAATTAAGGGCCAGCTCATTCCTCCCACTCATGATCTATAGAT CTATAGATCTCTCGTGGGATCATTGTTTTTCTCTTGATTCCCACTTTGTGGTTCTAAG TACTGTGGTTTCCAAATGTGTCAGTTTCATAGCCTGAAGAACGAGATCAGCAGCCT CTGTTCCACATACACTTCATTCTCAGTATTGTTTTGCCAAGTTCTAATTCCATCAGAA GCTAATTC TACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAG CAGCCCCGCTGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACA TTCCACATCCACCGGTAGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGC GCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCG CGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCA GATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGG CCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTG GGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCGAAGG TCCTCCCGAGGCCCGGCATTCTCGCACGCTTCAAAAGCGCACGTCTGCCGCGC TGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCGACCCGCTTAACA GCGTCAACAGCGTGCCGCAGATCTTGGTGGCGTGAAACTCCCGCACCTCTTC GGCCAGCGCCTTGTAGAAGCGCGTATGGCTTCGTACCCCTGCCATCAACACG CGTCTGCGTTCGACCAGGCTGCGCGTTCTCGCGGCCATAACAACCGACGTAC GGCGTTGCGCCCTCGCCGGCAACAAAAAGCCACGGAAGTCCGCCTGGAGCAG AAAATGCCCACGCTACTGCGGGTTTATATAGACGGTCCCCACGGGATGGGGA AAACCACCACCACGCAACTGCTGGTGGCCCTGGGTTCGCGCGACGATATCGT CTACGTACCCGAGCCGATGACTTACTGGCGGGTGTTGGGGGCTTCCGAGACA ATCGCGAACATCTACACCACACAACACCGCCTCGACCAGGGTGAGATATCGG CCGGGGACGCGGCGGTGGTAATGACAAGCGCCCAGATAACAATGGGCATGC CTTATGCCGTGACCGACGCCGTTCTGGCTCCTCATATCGGGGGGGAGGCTGG GAGCTCACATGCCCCGCCCCCGGCCCTCACCCTCATCTTCGACCGCCATCCCA TCGCCGCCCTCCTGTGCTACCCGGCCGCGCGATACCTTATGGGCAGCATGACC CCCCAGGCCGTGCTGGCGTTCGTGGCCCTCATCCCGCCGACCTTGCCCGGCAC AAACATCGTGTTGGGGGCCCTTCCGGAGGACAGACACATCGACCGCCTGGCC AAACGCCAGCGCCCCGGCGAGCGGCTTGACCTGGCTATGCTGGCCGCGATTC GCCGCGTTTATGGGCTGCTTGCCAATACGGTGCGGTATCTGCAGGGCGGCGG GTCGTGGCGGGAGGATTGGGGACAGCTTTCGGGGGCGGCCGTGCCGCCCCAG GGTGCCGAGCCCCAGAGCAACGCGGGCCCACGACCCCATATCGGGGACACGT TATTTACCCTGTTTCGGGCCCCCGAGTTGCTGGCCCCCAACGGCGACCTGTAT AACGTGTTTGCCTGGGCTTTGGACGTCTTGGCCAAACGCCTCCGTCCCATGCA TGTCTTTATCCTGGATTACGACCAATCGCCCGCCGGCTGCCGGGACGCCCTGC TGCAACTTACCTCCGGGATGGTCCAGACCCACGTCACCACCCCAGGCTCCAT ACCGACGATCTGCGACCTGGCGCGCACGTTTGCCCGGGAGATGGGGGAGGCT AACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGC AATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACG CGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCAT TGGGACCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCAACCCCCAAGTTC GGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAAGCCCTGCCAT AGCCACGGGCCCCGTGGGTTAGGGACGGGGTCCCCCATGGGGAATGGTTTAT GGTTCGTGGGGGTTATTATTTTGGGCGTTGCGTGGGGTCAGGTCCACGACTGG ACTGAGCAGACAGACCCATGGTTTTTGGATGGCCTGGGCATGGACCGCATGT ACTGGCGCGACACGAACACCGGGCGTCTGTGGCTGCCAAACACCCCCGACCC CCAAAAACCACCGCGCGGATTTCTGGCGCCGCCGGACGAACTAAACCTGACT ACGGCATCTCTGCCCCTTCTTCGCTGGTACGAGGAGCGCTTTTGTTTTGTATTG GTCACCACGGCCGAGTTTCCGCGGGACCCCGGCCAGGACCTGCAGAAATTGA TGATCTATTAAACAATAAAGATGTCCACTAAAATGGAAGTTTTTCCTGTCATA CTTTGTTAAGAAGGGTGAGAACAGAGTACCTACATTTTGAATGGAAGGATTG GAGCTACGGGGGTGGGGGTGGGGTGGGATTAGATAAATGCCTGCTCTTTACT GAAGGCTCTTTACTATTGCTTTATGATAATGTTTCATAGTTGGATATCATAATT TAAACAAGCAAAACCAAATTAAGGGCCAGCTCATTCCTCCCACTCATGATCT ATAGATCTATAGATCTCTCGTGGGATCATTGTTTTTCTCTTGATTCCCACTTTG TGGTTCTAAGTACTGTGGTTTCCAAATGTGTCAGTTTCATAGCCTGAAGAACG AGATCAGCAGCCTCTGTTCCACATACACTTCATTCTCAGTATTGTTTTGCCAA GTTCTAATTCCATCAGAAGCTAGCTT GGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGA AAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCG CCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAG TTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTAC GCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCT GCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCT GACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGC TGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC CGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTT AATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGG AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATA TGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA AAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTT TTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGA AAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAA CTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACG TTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTAT CCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCT CAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGA TGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATA ACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTA ACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTG GGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTA CTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAA AGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTG CTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATA CTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAA GATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGT TCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGAT CCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCT ACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGA AGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTG TAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATA CCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGT CGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAG CGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAAC GACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCA CGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTC GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCT TTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGT GATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCC TTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCC TGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAG CTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGC GAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTG GCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGG GCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCC CAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAG CGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAA Good luck, Chris Paszty 2nd e-mail: Dec 1995 Gene targeters: A couple of years ago I sent out the sequence compilations of the PGKneo and PGKtk cassettes that make up pPNT [Tybulewicz et al. Cell vol 65 p1153-1163 (1991)] with the goal of making it easier for people to construct targeting vectors using these 2 cassettes and to design screening strategies for identifying correctly targeted ES cell clones. The purpose of this follow up note is to address the issue of DNA rearrangement that can be encountered during vector construction. A good way to build one's targeting vector using pPNT has been to clone the short arm into the polylinker that is located between the PGKneo and PGKtk cassettes and to then clone the long arm into the unique XhoI site that lies on the other side of the PGKneo cassette. The final targeting vector can then be linearized at the unique NotI site. With regards to the effectiveness of negative selection by PGKtk in mammalian cells this configuration of the short and long arms as well as the linearization by NotI is ideal for two reasons. First, the distance between the PGKneo and PGKtk cassettes is minimized thereby making it more unlikely for events (such as endonuclease attack) other than homologous recombination to separate the two markers. Second, by linearizing with NotI the pUC/Bluescript region of the vector is left flanking the PGKtk cassette thereby decreasing the probability of PGKtk being inactivated by exonucleases. When building my first targeting vector I found that most (99%+) of the clones that were recovered after the last ligation step (cloning the long arm into the XhoI site) contained deletions and rearrangements. From talking to a number of people this seemed to be a fairly common occurrence when including both PGKneo and PGKtk in ones targeting construct. The unrearranged clone that I finally isolated turned out to be totally stable even when grown for many "passages". When miniprep DNA from this clone was retransformed into bacteria no rearrangement was observed. This seemed to indicate that the rearrangement problem was not due to the particular murine sequences that I had included in my targeting vector but was more likely due to the topology of the transforming DNA. Since I had used calf intestinal phosphatase (CIP) to dephosphorylate the XhoI ends of my XhoI cut short arm vector (to eliminate vector religation) the topology of DNA in my ligation mix was OC (open circular) containing dephosphorylated nicks. From the subsequent construction of a number of targeting vectors based on pPNT and a 2 tk version of pPNT (pPN2T) it is now clear that by not CIP treating the short arm vector during the cloning in of the long arm, the incidence of rearrangement is reduced to almost zero, thereby making it much easier to obtain the final targeting vector. With regards to vector religation without the CIP treatment one can still use the XhoI site for cloning in the long arm by playing with the insert-vector ratio to minimize vector religation. Alternatively, one could eliminate vector religation without using CIP and also get directional cloning of the long arm using the NotI and XhoI sites in pPNT, but because these two sites overlap in pPNT, achieving effective double digestion was thought to not be possible (93' NEB catalogue p 180). Recently the people at NEB (Moreira and Noren, Biotechniques 1995 vol 19 p56-59; Minimum Duplex Requirements for Restriction Enzyme Cleavage Near the Termini of Linear DNA Fragments) have reexamined the overlapping restriction enzyme site issue and their new results indicate that it should be possible to achieve effective double digestion of the NotI and XhoI sites in pPNT and pPNT derivatives by first cutting with NotI and then subsequently with XhoI. So although we haven't tried it , using NotI and XhoI may be a realistic option for the cloning in of the long arm. Another approach which we've been using successfully and prevents vector religation as well as allowing for forced directional cloning of the long arm is as follows: The recognition site for a fairly new 8 cutter, Sse8387I (CCTGCAGG, available from Amersham), is located between the NotI site and the PGKneo cassette in both pPNT, pPN2T and pPN2T-hGHterm. By double digesting the short arm-containing targeting vector with NotI and Sse8387I and then gel purifying it one can eliminate vector religation. The different cohesive ends also allow for the directional cloning of the long arm. There are various straightforward approaches for creating a NotI site and an Sse8387I site at either end of ones long arm: - if one is using PCR to obtain the long arm then it is easy to just include a NotI site on the 5' end of one of the primers and an Sse8387I site on the 5' end of the other primer. Cloning of the product leaves one with a long arm that is ready to clone into one's short arm-containing vector. - another approach involves creating a blue/white vector that has a blunt end cloning site in-between a NotI site and an Sse8387I site. The long arm can then be blunt end cloned in both orientations into this intermediate vector. The proper orientation clone leaves one with a long arm that is ready to clone into one's short arm-containing vector. To make a blue/white NotI-Sse8387I intermediate vector one can modify the polylinker of any common blue/white vector using custom oligos. Alternatively one can combine complementary fragments from two different blue/white vectors...the 1892 bp XbaI-AlwNI fragment from pUC18 (polylinker contains an Sse8387I site) and the 833 bp XbaI-AlwNI fragment from pBluescript SK +/- (polylinker contains a NotI site) can be combined to create such a vector (pUCNotSse). This hybrid blue/white vector has a HincII site (blunt end cutter) in-between the NotI site and the Sse8387I site. pUCNotSse polylinker: gagctccaccgcggtggcggccgctctagagtcgacctgcaggcatgcaagctt Over the past year, using the sequence compilations of PGKneo and PGKtk as well as the NotI-Sse8387I long arm cloning strategy, we've significantly increased the speed and ease with which we build our targeting vectors. Hope this information is useful to others. Chris Paszty Lawrence Berkeley Laboratory At Amgen Inc. as of Feb 1998 cpaszty@amgen.com