Isolation and characterisation of tilapia beta-actin promoter and comparison of its activity with carp beta-actin promoter

The regulatory sequence including proximal promoter, untranslated exon 1 and intron 1 of the beta-actin gene from tilapia (Oreochromis niloticus) has been isolated and spliced to a beta-galactosidase reporter gene to test its activity. Comparisons of promoter activity have been carried out with three different constructs: (1) 1.6 kb tilapia beta-actin regulatory sequence, (2) 1.5 kb carp beta-actin regulatory sequence, and (3) 4.7 kb carp beta-actin regulatory sequence. Although the 1.6 kb tilapia beta-actin regulatory sequence gave slightly different expression patterns in tilapia embryos assayed by in situ X-gal staining, no difference was observed in expression level when the tilapia sequence was compared with the 4.7 kb carp beta-actin regulatory sequence by quantitative assay. In comparison with the 1.5 kb carp beta-actin regulatory sequence, the 1.6 kb tilapia beta-actin regulatory sequence gave higher expression levels in tilapia embryos, while a reverse result was observed in zebrafish embryos. In cell transfection experiments, the 1.6 kb tilapia beta-actin regulatory sequence showed three to four times better activity in blue gill cells than either the 4.7 kb carp beta-actin or the 1.5 kb carp beta-actin regulatory sequences. The 1.6 kb tilapia beta-actin regulatory sequence also drove higher reporter gene activity in somatic cells of tilapia than did the 4.7 kb carp beta-actin regulatory sequence following direct injection of constructs into muscle. Therefore, taken together, the data demonstrate that the tilapia beta-actin promoter can be used as an efficient regulatory sequence to produce autotransgenic tilapia.     

Comparison of the activity of carp and rat β-actin gene regulatory sequences in tilapia and rainbow trout embryos

A comparative study on the level of expression of lacZ reporter constructs driven by equivalent carp and rat β-actin regulatory sequences was carried out in embryos of tilapia and rainbow trout. DNA was microinjected into fertilised tilapia and rainbow trout eggs and the embryos/fry were assayed at various developmental stages for β-galactosidase expression. We provide evidence to demonstrate that the carp β-actin promoter/lacZ reporter gene is expressed at higher levels than the equivalent rat β-actin construct in both species. © 1996 Wiley-Liss, Inc.     

Targeting Myofibroblasts in Model Systems of Fibrosis by an Artificial α-Smooth Muscle-Actin Promoter Hybrid

Myofibroblasts are the main cell types producing extracellular matrix proteins in a variety of fibrotic diseases. Therefore, they are useful targets for studies of intracellular communication and gene therapeutical approaches in scarring diseases. An artificial promoter containing the −702 bp regulatory sequence of the α-smooth muscle actin (SMA) gene linked to the first intron enhancer sequence of the β-actin gene and the β-globin intron-exon junction was constructed and tested for myofibroblast-dependent gene expression using the green fluorescent protein as a reporter. Reporter expression revealed myofibroblast-specific function in hepatic and renal myofibroblasts, in vitro. In addition, differentiation-dependent activation of the SMA-β-actin promoter hybrid was shown after induction of myofibroblastic features in mesangial cells by stretching treatment. Furthermore, wound healing experiments with SMA-β-actin promoter reporter mice demonstrated myofibroblast-specific action, in vivo. In conclusion, the −702 bp regulatory region of the SMA promoter linked to enhancing β-actin and β-globin sequences benefits from its small size and is suggested as a promising tool to target myofibroblasts as the crucial cell type in various scarring processes.     

Electron Microscopic Localization of LacZ Expression in the Proximal Convoluted Tubular Cells of the Kidney in Transgenic Mice Carrying Chimeric Erythropoietin/lacZ Gene Constructs

Regulated expression of the erythropoietin (EPO) gene in the adult kidney plays a key role in the regulation of erythropoiesis. However, uncertainty exists regarding the type of kidney cells involved in EPO gene expression. We previously showed by light microscopy that the lacZ reporter gene is expressed and inducible by hypoxia/anemia in the proximal convoluted tubular (PCT) cells of the kidneys of transgenic mice carrying the 5′-lacZ construct, in which the lacZ gene was placed downstream of a 7.0-kb mouse EPO gene segment containing 6.5 kb of the 5′-flanking sequence. We, report here the light and transmission electron microscopic examination of lacZ expression in the kidneys of transgenic mice carrying the 5′-lacZ construct and two additional constructs carrying the 6.5-kb 5′-flanking sequence with the body of the gene alone, or along with the 1.2-kb 3′-flanking sequence. The electron microscopic analyses unequivocally demonstrated that lacZ under the regulatory control of the 6.5-kb 5′-flanking sequence with or without the body of the gene and the 1.2-kb 3′-flanking sequence was expressed predominantly in the proximal convoluted tubular cells of the kidney following hypoxia induction.     

Denitrification enzymes of Bacillus stearothermophilus

Abstract A hybrid trpPO:lacO regulatory sequence was cloned upstream of a promoterless lacZ gene and recombined onto a Λ bacteriophage. Escherichia coli lysogens representing the four possible phenotypes for lacI and trpR were constructed and the synthesis of β -galactosidase was assayed under various growth conditions. The results illustrated that both control elements could be efficiently and independently regulated by the addition or omission of appropriate accessory molecules.     

Insertion of a 27 amino acid viral peptide in different zones of Escherichia coliβ-galactosidase: Effects on the enzyme activity

Abstract Seven internal, putatively exposed regions of Escherichia coli β-galactosidase have been explored regarding their tolerance to insertions of large foreign peptides. Small sequence modifications, including amino acid substitutions and small deletions, were introduced into the lacZ gene to generate unique Bam HI restriction sites. By using these mutant genes, a 27 amino acid stretch reproducing the hypervariable loop of foot-and-mouth disease virus VP1 protein (site A) was further inserted in predefined regions of the enzyme. Among the 13 resulting engineered proteins only three, carrying sequence modifications within a short region, are active, with only moderate reduction of their specific activities. The identified permissive region, which involves amino acids 275 to 279, seems to be a flexible area that could be appropriate incorporate and study biological properties of heterologous peptides in correctly folded β-galactosidase chimeric proteins.     

Engineering lacZ Reporter gene into an ephA8 bacterial artificial chromosome using a highly efficient bacterial recombination system

In this report, we describe an optimized method for generation of ephA8 BAC transgenic mice expressing the lacZ reporter gene under ephA8 regulatory sequences. First, we constructed a targeting vector that carries a 1.2 kb ephA8 DNA upstream of its first exon, a lacZ expression cassette, a kanamycin cassette, and a 0.7 kb ephA8 DNA downstream of its first exon. Second, the targeting vector was electroporated into cells containing the ephA8 BAC and pKOBEGA, in which recombinases induce a homologous recombination between the ephA8 BAC DNA and the targeting vector. Third, the FLP plasmid expressing the Flipase was electroporated into these bacteria to eliminate a kanamycin cassette from the recombinant BAC DNA. The appropriate structures of the modified ephA8 BAC DNA were confirmed by Southern analysis. Finally, BAC transgenic mouse embryos were generated by pronuclear injection of the recombinant BAC DNA. Whole mount X-gal staining revealed that the lacZ reporter expression is restricted to the anterior region of the developing midbrain in each transgenic embryo. These results indicate that the ephA8 BAC DNA contains most, if not all, regulatory sequences to direct temporal and spatial expression of the lacZ gene in vivo.     

The Fugu rubripes tyrosinase gene promoter targets transgene expression to pigment cells in the mouse

The regulation of the mouse tyrosinase gene expression is controlled by a highly conserved element at -100 bp, the M-box, and an enhancer at -12 kb. In most vertebrates, the length of intergenic sequences makes it difficult to analyze the whole gene and the complete regulatory region. We took advantage of the compact Fugu genome to identify regulatory regions involved in pigment cell-specific expression. We isolated the Fugu tyrosinase gene, and identified putative cis-acting regulatory elements within the promoter. We then asked whether the Fugu promoter sequence functions in mouse pigment cells. We showed that E11.5 transgenic embryos bearing 6 kb or 3 kb of Fugu tyrosinase 5' sequence fused to the reporter gene lacZ revealed melanoblast and RPE-specific expression. This is the first evidence that the tyrosinase promoter is active at midgestation in melanoblasts, long before the onset of pigmentation.     

Fish transgene expression by direct injection into fish muscle.

The ability of a promoter sequence to drive expression of a reporter gene can be determined by direct injection of copies of the cloned sequence into fish muscle, followed by biopsy of muscle from the site of injection. We describe a set of experiments in which copies of the constructs FV1 and FV2, both comprising a carp beta-actin promoter sequence spliced to the bacterial reporter gene CAT, were injected into the muscle of tilapia fish )Oreochromis niloticus) of between 5 and 8 cm body length. The site of injection was carefully determined so that biopsy samples could be recovered from the injection site 24 hours, 48 hours, and 7 days after injection. Biopsy samples of muscle were homogenized and used for CAT assays. CAT activity was successfully detected in many of the muscle samples.     

Copy Number Related Transgene Expression and Mosaic Somatic Expression in Hemizygous and Homozygous Transgenic Tilapia (Oreochromis Niloticus)

Three lines of transgenic tilapia (Oreochromisniloticus) fish were generated with a constructcontaining a lacZ reporter gene spliced to a 4.7kb 5 regulatory region of a carp beta actin gene. All these three lines contain different copy numbers oftransgenes and the levels of lacZ expressionwere found to be related to transgene copy number.Mosaic patterns of somatic lacZ expression wereobserved in these three lines which differed between linesbut were consistent within a line. We also observedthat expression of the reporter gene in homozygoustransgenic fish was approximately two-fold greater thanin the hemizygous transgenics. Analysis of expressionof the reporter gene on a tissue-to-tissue basisdemonstrated that lacZ expression of thereporter gene in stably transformed fish occured withvariable intensity in different organs and tissues andwas also sometimes variable in different cells of thesame tissue in G1and G2 generations of the transgenic lines.