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PubMed2005

Magnetic resonance imaging and characterization of spontaneous lesions in a transgenic mouse model of tuberous sclerosis as a model for endothelial cell-based transgene delivery.

Tuberous sclerosis (TSC) is an autosomal dominant genetic disorder characterized by abnormalities in cellular migration, proliferation, and differentiation in many tissues. Benign hamartomas develop in multiple organs, believed to be caused by somatic mutation in addition to germ line mutation to cause loss of both alleles of either the TSC1 or TSC2 tumor suppressor gene, with resultant dysregulated growth due to loss of hamartin or tuberin function, respectively. This study focuses on detecting spontaneous lesions in a knockout mouse model of TSC2 by magnetic resonance imaging (MRI) and exploring the efficiency of introducing gene products into lesions, using transduced endothelial cells as gene vehicles. MRI was shown to be effective in detecting spontaneous lesions in multiple tissues as a means of assessing the prevalence of tumors. Tsc(2+/) heterozygous mice were screened at 12-24 months of age. MRI detected 100% of the renal lesions (cystadenomas, renal cell carcinomas) and 75% of the hepatic lesions (hemangiosarcomas), later identified by histology. Cell-mediated gene delivery was evaluated by immunohistochemical analysis of renal, hepatic, and lung lesions after intravenous delivery of MS1 mouse endothelial cells, transduced to express an enhanced form of green fluorescent protein (EGFP). Preliminary immunohistochemical analysis, using a polyclonal antibody to EGFP and a horseradish peroxidase-diaminobenzidine detection system, revealed these cells throughout liver, kidney, and lung sections from injected animals, organs that are frequently affected in TSC2 patients, as well as within the lesions themselves.

Animals↗
PubMed2006

Transgenic wine yeast technology comes of age: is it time for transgenic wine?

Saccharomyces cerevisiae is the main yeast responsible for alcoholic fermentation of grape juice during wine making. This makes wine strains of this species perfect targets for the improvement of wine technology and quality. Progress in winemaking has been achieved through the use of selected yeast strains, as well as genetic improvement of wine yeast strains through the sexual and pararexual cycles, random mutagenesis and genetic engineering. Development of genetically engineered wine yeasts, their potential application, and factors affecting their commercial viability will be discussed in this review.

Fermentation↗
PubMed2001

The poplar bark storage protein gene (Bspa) promoter is responsive to photoperiod and nitrogen in transgenic poplar and active in floral tissues, immature seeds and germinating seeds of transgenic tobacco.

In Populus, seasonal nitrogen storage involves the accumulation of a 32 kDa bark storage protein (BSP) in the inner bark parenchyma and xylem rays. Poplar BSPs are encoded by a multigene family and one member, bspA, has been cloned and sequenced. The regulation of bspA was investigated by transforming either hybrid poplar or tobacco with a chimeric gene consisting of the 2.8 kb bspA promoter fused to the coding region of beta-glucuronidase (uidA). In transformed poplar, the bspA 2.8 kb promoter conferred both short-day (SD) and nitrogen (N) inducibility to GUS and activity was localized to the bark (primary and secondary phloem, and cortex) and xylem rays. Night-break treatments inhibited SD induction of GUS. Deletion of the 1.6 kb distal DNA sequences from the bspA promoter eliminated SD induction of GUS while some N induction was retained. These results indicate that although poplar BSP is encoded by a multigene family, transcriptional activation of bspA per se can account for bsp expression in bark and xylem rays in response to either SD or N treatment. These results also show that the elements responsible for SD or N induction are separable. Because of the long generation intervals associated with trees, the developmental regulation of bspA in flowers, developing seeds, and germinating seeds was investigated by transforming the 2.8 kb bspA-promoter::uidA chimeric gene into tobacco. The bspA promoter was active in developing tobacco floral tissues and in seeds during early stages of embryogenesis, decreased progressively during seed maturation and regained activity upon seed germination. Although seed storage proteins of poplar share some similarities to poplar BSP, the observed developmental expression patterns in tobacco are consistent with a role for bspA in vegetative rather than seed storage protein storage.

Gene Expression Regulation, Developmental↗
PubMed1996

[Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. II. Transgenic tobacco plants expressing the bacterial beta-glucanase gene from Clostridium thermocellum,--a model for studying the differential expression of stress response-related genes].

The modified hybrid beta-1,3-glucanase gene (glc) of Clostridium thermocellum was expressed in tobacco Nicotiana tabacum. The glc gene was cloned into two plasmids, pC27-glc and pC29-glc, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter of Arabidopsis, respectively. These constructions were used for transformation of agrobacteria followed by transfer into plants. In transformed plants, each plasmid caused a high level of activity of thermostable bacterial glucanase not observed in reference plants. The plants obtained were used to study activation of some defense-related genes induced by their interaction with either tobacco mosaic virus (TMV) or a pathogenic fungus.

Cloning, Molecular↗
PubMed1998

An agent that increases tumor suppressor transgene product coupled with systemic transgene delivery inhibits growth of metastatic lung cancer in vivo.

Low levels of gene expression following systemic delivery have impaired the effectiveness of tumor suppressor gene replacement in treating metastases. We asked whether combined treatment with 2-methoxyestradiol (2-Me), which increases levels of wild-type p53 protein in cancer cells, and the systemic administration of an adenoviral vector expressing wild-type p53 (Ad-p53) would inhibit the growth of human metastatic lung cancer cells in vivo. The simultaneous administration of p53 and 2-Me resulted in a greater than additive reduction with the lung colony count reduced to 33% of its control value. These results suggest that the synergistic effect of 2-Me and Ad-p53 in combination treatment may have application in the systemic treatment of cancer.

2-Methoxyestradiol↗
PubMed2001

Growth hormone and fertility in oMt1a-oGH transgenic mice.

Female mice carrying a regulatable growth hormone transgene (oMt1a-oGH) are subfertile when the transgene is actively expressed. This study was designed to characterize subfertility caused by increased concentrations of growth hormone. In particular, this study aimed to: (i) determine the effects of transgene activation and inactivation on mating, conception, maintenance of pregnancy, ovulation rate, litter characteristics and embryonic survival at day 17 of pregnancy, (ii) characterize oestrous cyclicity in transgenic versus wild-type female mice, and (iii) correlate corticosterone concentrations with transgene expression and reproductive performance. Transgenic and wild-type female mice were allocated randomly to one of four treatment groups at weaning: (i) transgenic female mice that always express the transgene, (ii) transgenic female mice that never express the transgene, (iii) transgenic female mice that express the transgene for up to 8 weeks of age and (iv) non-transgenic wild-type female mice receiving the transgene stimulus until 8 weeks of age. Activation followed by inactivation of the transgene resulted in an increased incidence of remating, resulting in an extended interval to establish pregnancy in comparison with all other treatment groups. Transgenic mice that always expressed the transgene and those that expressed the transgene for up to 8 weeks of age had lower pregnancy rates and higher ovulation rates compared with mice from other treatment groups. Both embryonic survival and the duration of the oestrous cycle did not differ among treatment groups. Active expression of the transgene resulted in an increase in the plasma concentration of corticosterone, which was associated with reduced fertility. These data indicate that the presence of a high growth hormone concentration impedes the establishment and maintenance of pregnancy. Increased plasma corticosterone concentrations may interfere with implantation as well as potentiate leptin resistance, which has been reported previously in studies with these mice.

Analysis of Variance↗
PubMed2002

Production of transgenic medaka with increased resistance to bacterial pathogens.

Cecropins, first identified in silk moth (Hyalophora cecropia), are a group of antimicrobial peptides with bactericidal activity against a broad spectrum of bacteria. In this study we investigated whether (1) this group of antimicrobial peptides could exhibit bactericidal activity toward known fish bacterial pathogens and (2) expression of cecropin transgenes in transgenic medaka (Oryzias latipas) could result in increasing resistance of the transgenic fish to infection by fish bacterial pathogens. Cecropin gene construct containing silk moth preprocecropin B, procecropin B and cecropin B, and porcine cecropin P1 driven by a cytomegalovirus (CMV) promoter were transfected into chinook salmon embryonic cells (CHSE-214) by lipofection, and the resulting permanent transformants were collected. In an "inhibition zone" assay medium isolated from each transformant exhibited strong bactericidal activity toward known fish bacterial pathogens such as Pseudomonas fluorescens, Aeromonas hydrophila, and Vibrio anguillarum. The same cecropin transgene constructs were introduced into newly fertilized medaka eggs by electroporation to produce transgenic fish. About 40% to 60% of the embryos survived from electroporation, and about 5% to 11% of the surviving fish were shown to contain cecropin transgenes by polymerase chain reaction analysis of genomic DNA samples isolated from presumptive transgenic fish. These P1 transgenic fish were used as founder stocks, and following generations of successive breeding, a total of 20 F2 families of transgenic fish were established. Expression of cecropin transgenes was detected in the F2 transgenics by reverse transcriptase polymerase chain reaction analysis. Southern blot analysis of genomic DNA isolated from different F2 fish showed that cecropin transgenes were integrated into the genomes of F2 transgenic fish. To determine whether transgenic fish carrying cecropin transgenes could exhibit resistance to infection by known fish bacterial pathogens, F2 transgenic fish from different families and control fish were challenged with P. fluorescens and V. anguillarum at a 60% lethal dose. Challenge studies showed that while about 40% of the control fish were killed by both pathogens, only up to 10% of the F2 transgenic fish were killed by P. fluorescens and about 10% to 30% by V. anguillarum. These results clearly showed that the transgenic medaka carrying cecropin transgenes had acquired elevated resistance to bacterial infection.

Journal Article↗