Cre is a 38-kDa recombinase protein from bacteriophage P1 that catalyzes reciprocal site-specific recombination between 34-base-pair (bp) loxP sites (2). In bacteria and in vitro Cre mediates both intramolecular (excisive or inversional) and intermolecular (integrative) recombination at loxP sites. Surprisingly, Cre also promotes both excisive (3-5) and integrative (6) recombination in eukaryotic cells.To select targeted integration in a wide range of mammalian cells, we have designed an inactive lox-neo fusion gene to use as a lox chromosomal target. Cre-mediated integration of a promoter-ATG-lox targeting vector into the chromosomal target reconstructs a functional ATG-lox-neo fusion gene and renders cells resistant to the neomycin analogue G418. We constructed CHO cell derivatives with a single randomly placed genomic copy of this lox target and then characterized Cre-mediated targeting in these cells. To monitor gene expression, we placed a f-actin-lacZ reporter gene into the lox targeting vector, isolated a number of independent targeted lines, and measured the resulting level of ,-galactosidase. MATERIALS AND METHODSPlasmids and DNA Constructions. Plasmids pBS185 (6), pBS74 (5), and pRH43 (7) have been described. The defective lox-neo gene fusion plasmid pSFl (Fig. 1A) was constructed in a number of steps. First, the 2.4-kilobase (kb) EcoRIBamHI fragment of pRH43 containing loxP and the ampicillin-resistance (Ap) gene was ligated to the 2.8-kb EcoRI-Bgl II neo fragment from pBS74. The first five codons of the neo gene were removed and the neo coding region was fused in-frame to the loxP site by PCR overlap mutagenesis using synthetic primers (8), to give pSF1. The resulting product was sequenced and is shown in Fig. 1A. Because the N terminus of neomycin phosphotransferase is nonessential (9), we expected that provision of a translational start signal to the pSF1 construct (by Cre-mediated recombination; see below) would result in a functional lox-neo fusion protein. The 2.6-kb Chinese hamster DHFR minigene from pMG1 (10) was cloned into the BamHI site of pSF1 to generate pSF14 (Fig. 1A).The targeting vector pBS226 (Fig. 1B) was constructed by digesting pUC18 with Asp718, filling in the cohesive ends, and religating the DNA. Synthetic oligonucleotides were inserted into the polylinker to generate (i) a new AUG start having optimized eukaryotic and prokaryotic translational start signals, and (ii) a novel loxGI C2 site with a Sph I site at one end. Finally, the 0.8-kb Xba I hCMV fragment from pBS185 was inserted to give pBS226. The human /3-actin promoter-lacZ cassette (fusion is at position 52 of the /3-actin sequence) with the simian virus 40 polyadenylylation site was obtained on a 3.95-kb BamHI-Bgl II fragment (from J.Rossant, University of Toronto, through R. Scott, DuPont) and was cloned into the BamHI site of pBS226 in both orientations to give pSF18 and pSFl9 (Fig. 1B). The 600-bp promoter lacks both the upstream region and the enhancercontaining intervening sequence (11).Cell Lines and Gene...

With v-yes DNA as the probe, a human cDNA library made from placental RNA was screened under relaxed conditions, and DNA clones derived from a novel genetic locus, termed lyn, were obtained. Nucleotide sequencing revealed that lyn could encode a novel tyrosine kinase that was very similar to mouse T-lymphocyte-specific tyrosine kinase p56lck and the v-yes protein as well as to the gene products of v-fgr and v-src. Northern hybridization analysis revealed that a 3.2-kilobase lyn mRNA was expressed in a variety of tissues of the human fetus. The pattern of lyn mRNA expression was different from those of related genes, such as yes and syn. Hybridization analysis of DNA from sorted chromosomes showed that the lyn gene is located on human chromosome 8 q13-qter.

We have used comparative genomic hybridization (CGH) to survey genomic regions with aberrant copy numbers of DNA sequences in pancreatic adenocarcinoma. In 12 cell lines and 6 primary tumors from 18 patients with pancreatic adenocarcinomas, highly frequent losses (>60%) were observed on chromosome arms 6q, 9p, and 18q and the Y chromosome. Moderately frequent losses (40–60%) were observed on chromosome arms 3p, 4q, 8p, and 21q. Interestingly, these samples showed extremely high frequencies of increases in copy numbers of DNA sequences on the long arm of chromosome 20 (15/18, 83%). We further analyzed five cell lines by fluorescence in situ hybridization (FISH) with probes on chromosome 20 to define the increase in copy number more accurately, and we found that 20q was increased to between 5 and 8 copies per cell. These results suggest the existence of an oncogene or oncogenes on 20q that play a role in the development and/or the progression of pancreatic carcinogenesis. Genes Chromosom. Cancer 19:161–169, 1997. © 1997 Wiley‐Liss Inc.

Epigenetic silencing through methyl-CpG (mCpG) is implicated in many biological patterns such as genome imprinting, X chromosome inactivation, and cancer development. In this process, the mCpG binding domain (MBD) proteins play an essential role in transmitting epigenetic information to downstream regulatory proteins. Among the five MBD proteins identified so far, MBD4 has been the only exception; it has long been thought to be a DNA repair protein. Herein we demonstrate that MBD4 has the ability to repress transcription through mCpG. Transcriptional repression by the MBD4 is histone deacetylase (HDAC) dependent, and MBD4 directly binds to Sin3A and HDAC1 at three central regions that overlap transcriptional repression domains. Furthermore, a chromatin immunoprecipitation assay clearly shows that MBD4 binds to hypermethylated promoters of the p16INK4a and hMLH1 genes. These results suggest that MBD4 is one of the essential components involved in epigenetic silencing in cancer and its repair activity is necessary for the maintenance of hypermethylated promoters.

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DUSP6 (alias PYST1), one of the dual-specificity tyrosine phosphatases, is localized on 12q21, one of the regions of frequent allelic loss in pancreatic cancer. This gene is composed of three exons, and two forms of alternatively spliced transcripts are ubiquitously expressed. Although no mutations were observed in 26 pancreatic cancer cell lines, reduced expressions of the full-length transcripts were observed in some cell lines, which may suggest some role for DUSP6 in pancreatic carcinogenesis.

Initiation and progression of human caner not only depends on genetic alterations but also on epigenetic changes such as DNA methylation and histone modifications. Aberrant DNA hypermethylation in the promoter regions of genes is the most well-defined epigenetic change in tumors and is associated with inappropriate gene silencing. This feature can be utilized to search for tumor-specific DNA methylation biomarkers and to examine candidate DNA biomarkers for clinical use. DNA methylation biomarker is defined as a molecular target that undergoes DNA methylation changes in carcinogenesis. Such a biomarker is useful for early detection of cancer, predicting and/or monitoring the therapeutic response, and detection of recurrent cancer. In this review, we describe the mechanism that establishes and maintains DNA methylation patterns as well as the mechanism of aberrant gene silencing in cancer, and then we introduce methods to isolate the DNA methylation biomarkers. We also summarize the current status of clinical implementation for some of the most widely studied and well-validated DNA methylation biomarkers, including tissue factor pathway inhibitor 2 (TFPI2), septin 9 (SEPT9), glutathione S-transferase pi 1 (GSTP1), and O 6 -methylguanine-DNA methyltransferase (MGMT), and assess the clinical potential of these biomarkers for risk assessment, early diagnosis, prognosis, treatment, and the prevention of cancer. Finally we describe the possible involvement of 5-hydroxymethylcytosine in cancer; this is a recently discovered 5-methylcytosine oxidation derivative and might have a diagnostic potential in certain cancers. Abnormal DNA methylations are leading candidates for the development of specific markers for cancer diagnosis and therapy.