Stable integration of large (>100 kb) PAC constructs in HaCaT keratinocytes using an integrin-targeting peptide delivery system.

Transfer of large DNA constructs in gene therapy studies is being recognised for its importance in maintaining the natural genomic environment of the gene of interest and providing tissue-specific regulation and control. However, methods used to deliver such constructs have been poorly studied. We used a receptor-mediated, integrin-targeting transfection system enhanced by liposomes, to deliver a 110 kb PAC (P1-based artificial chromosome) to HaCaT keratinocytes. The PAC contained the collagen VII locus, an EGFP (enhanced green fluorescent protein) reporter gene and the puromycin resistance gene (pac) to allow selection of stably transfected cells. Analysis of puromycin resistant and EGFP-expressing colonies by Western blot showed that collagen VII production increased dramatically after transfection, indicating successful transfer of a large fully functional genomic locus. Fluorescent in situ hybridisation (FISH) and Southern blot analysis revealed that the PAC had integrated as at least one copy per cell. EGFP expression has persisted for 35 weeks, suggesting stable transgene expression. We conclude that the integrin-targeting peptide method of gene delivery is an effective means of stably delivering large DNA constructs to human keratinocytes and could be of benefit for genomic gene therapy approaches.

The cholinergic locus: ChAT and VAChT genes.

The gene encoding the vesicular acetylcholine transporter has been localized within the first intron of the gene encoding choline acetyltransferase and is in the same transcriptional orientation. These two genes, whose products are required for the expression of the cholinergic phenotype, could therefore be coregulated. The promoters of both genes have been identified. The mechanisms that account for the regulation of the expression of both genes are now being investigated.

Specific vesicular acetylcholine transporter promoters lie within the first intron of the rat choline acetyltransferase gene.

The sequence encoding the vesicular acetylcholine transporter (VAChT) has recently been localized within the first intron of the choline acetyltransferase (ChAT) gene in various species. In rat, we previously identified a class of VAChT mRNAs that may originate from the same promoter region as two ChAT mRNAs. Here, we demonstrate by a detailed analysis of the 5'-noncoding region of the VAChT gene, that two specific VAChT promoters lie within the first intron of the ChAT gene. Two VAChT mRNAs are generated from these promoters. These results demonstrate that the promoter regions of these two genes are intermingled, which highlight the unique organization of the ChAT/VAChT gene locus.

Coregulation of two embedded gene products, choline acetyltransferase and the vesicular acetylcholine transporter.

The gene encoding the vesicular acetylcholine transporter (VAChT) has recently been localized within the first intron of the gene encoding choline acetyltransferase (ChAT) and is in the same transcriptional orientation. These two genes, whose products are required for the expression of the cholinergic phenotype, could therefore be coregulated. We thus tested the effects on VAChT gene expression of the cholinergic differentiation factor/leukemia inhibitory factor and retinoic acid, both of which induce ChAT activity and increase ChAT mRNA levels in cultured sympathetic neurons. These factors increased both the number of binding sites for vesamicol, a specific ligand of VAChT, and VAChT immunoreactivity. This increase in the number of VAChT molecules resulted from an increase in the amount of VAChT mRNA, as assessed by reverse transcription-PCR and which paralleled that of ChAT mRNAs. These data suggest a functional role for ChAT and VAChT gene organization and are consistent with the existence of a coregulatory mechanism for the embedded ChAT and VAChT genes.

A unique gene organization for two cholinergic markers, choline acetyltransferase and a putative vesicular transporter of acetylcholine.

Choline acetyltransferase (ChAT) is the biosynthetic enzyme of acetylcholine. In mammalian tissues, it is encoded by multiple mRNAs with different 5'-ends. This diversity results from the alternative usage of three promoters and from differential splicing events. Here, we show that the first intron of the rat ChAT gene contains an open reading frame that encodes a potential vesicular acetylcholine transporter based on the following criteria. (i) The encoded protein is structurally similar to transporter proteins, the highest identity being found with the vesicular acetylcholine transporters from Torpedo and Caenorhabditis elegans (77 and 56%, respectively, in 352 amino acids). (ii) The corresponding mRNAs exhibit a cholinergic expression profile. Amplification experiments with spinal cord cDNA revealed that at least three mRNAs encode this transporter. Two contain the same 5' non-coding region as two ChAT mRNAs and, therefore, are derived from the ChAT transcription unit by alternative splicing. The third mRNA may be transcribed from an additional internal promoter.

Regulation by CDF/LIF and retinoic acid of multiple ChAT mRNAs produced from distinct promoters.

The cholinergic differentiation factor/leukaemia inhibitory factor (CDF/LIF) and retinoic acid (RA) induce in sympathetic neurones, a switch from the noradrenergic to the cholinergic neurotransmitter phenotype. In particular, these molecules alter the activities of the biosynthetic enzymes choline acetyltransferase (ChAT), tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). Recently, five rat ChAT mRNA species have been identified although no data have yet been reported concerning their production and regulation in sympathetic neurones. By use of the reverse transcription polymerase chain reaction technique we analysed the effects of CDF/LIF and RA on the levels of ChAT, TH and DBH mRNAs. Each ChAT mRNA was produced in sympathetic neurones and was induced by both molecules, whereas the mRNAs encoding TH and DBH enzymes were down-regulated.

Isolation and sub-chromosomal localization of a DNA fragment of the human choline acetyltransferase gene.

A DNA fragment of 219 bp was obtained by polymerase chain reaction (PCR) on human genomic DNA using two oligonucleotide mixtures derived from peptide sequences of human placenta choline acetyltransferase (ChAT) and from partially conserved amino acid sequences of rat, porcine and Drosophila ChAT. Sequence homology with porcine ChAT demonstrated that this fragment is part of the human ChAT gene. This gene was assigned to chromosome 10 by hybridization of the 219 bp DNA probe with DNA from human-hamster somatic cell hybrids, and to region 10q11.2-10qter by PCR experiments.

Epidermal growth factor-induced phosphoinositide hydrolysis. Modulation by protein kinase C.

A short-term treatment with phorbol 12,13-dibutyrate (PDBu) was found to inhibit totally the epidermal growth factor (EGF)-stimulated phosphoinositide hydrolysis in A431 cells, whereas long-term pretreatment with PDBu, which is known to down regulate protein kinase C, induced a greater accumulation of the EGF-triggered inositol phosphate accumulation, particularly of Ins(1,3,4,5)P4. The increased Ins(1,4,5)P3/Ins(1,3,4,5)P4 formation in the PDBu long-term pretreated cells was coincident with the increased Ca2+ influx stimulated by EGF in the same cells. Since long-term pretreatment with PDBu was found to enhance the EGF signals, an explanation for the synergism between EGF and phorbol esters in the induction of DNA synthesis is provided.