Transgenic mice engineered to target Cre/loxP-mediated DNA recombination into catecholaminergic neurons

To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, we generated transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter. Immunohistochemistry performed on transgenic mouse brain sections revealed a high number of cells expressing Cre in areas where TH is normally expressed, including the olfactory bulb, hypothalamic and midbrain dopaminergic neurons, and the locus coeruleus. Double immunohistochemistry and immunofluorescence indicated that colocalization of TH and Cre is greater than 80%. Cre expression was also found in TH-positive amacrine neurons of the retina, chromaffin cells of the adrenal medulla, and sympathetic ganglia. We crossbred TH-Cre mice with the floxed reporter strain Z/AP and observed efficient Cre-mediated recombination in all areas expressing TH, indicating that transgenic Cre is functional. Therefore, we have generated a valuable transgenic mouse strain to induce specific mutations of "floxed" genes in catecholaminergic neurons.     

Effect of heavy metals on the uptake of [3H]-L-histidine by the polychaete Nereis succinea

Integumentary uptake of [3H]-L-histidine by Nereis succinea was measured in the presence and absence of selected heavy metals and the amino acid L-leucine in 60% artificial seawater (ASW). The time course of 10 microM [3H]-L-histidine uptake into worms over a 60 min incubation was approximately doubled in the presence of 0.5 microM zinc and when calcium in the incubation medium was reduced from 6 mM to 5 microM the stimulatory effect of zinc on amino acid accumulation was reduced and uptake under the latter conditions was approximately half that of the control. Zinc stimulation of [3H]-L-histidine influx was a hyperbolic function of zinc concentration over the range 0 to 50 microM metal and displayed an apparent activation or affinity constant of 385+/-127 nM Zn(2+). The hyperbolic stimulatory effect of 1 microM Zn(2+) on the time course of 10 microM [3H]-L-histidine uptake was abolished in the presence of 25 microM L-leucine, suggesting that this amino acid shared the same transport system as [3H]-L-histidine and acted as a potential competitive inhibitor. Influx of [3H]-L-histidine was a hyperbolic function of external amino acid concentration and displayed an apparent affinity constant (Km) of 23.71+/-5.02 microM and an apparent aximal velocity (J(max)) of 4701+/-449 pmol/g dry wt.x15 min. Addition of 0.5 microM zinc resulted in a four-fold increase in J(max) and a doubling of K(m), suggesting the effect of the metal was mostly on the rate of amino acid transport. [3H]-L-histidine influx was mildly stimulated by Fe(2+) (0.5 microM), but was unaffected by either Ag(+) or Al(3+) (both at 0.5 microM). These results suggest that [3H]-L-histidine uptake into worm integument may take place by the classical Na(+)-independent L-transport system shared by L-leucine and regulated by exogenous calcium and other divalent metal concentrations.     

Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types

The muscle creatine kinase (MCK) gene is expressed at high levels only in differentiated skeletal and cardiac muscle. The activity of the cloned enhancer–promoter has previously been shown to be dependent on the Trex element which is specifically bound by a yet unidentified nuclear factor, TrexBF. We have further characterized the function of the Trex site by comparing wild-type and Trex-mutated MCK transgenes in five mouse skeletal muscles: quadriceps, extensor digitorum longus (EDL), soleus, diaphragm, and distal tongue, as well as in heart ventricular muscle. Several types of statistical analysis including analysis of variance (ANOVA) and rank sum tests were used to compare expression between muscle types and between constructs. Upon mutation of the Trex site, median transgene expression levels decreased 3- to 120-fold in the muscles examined, with statistically significant differences in all muscles except the EDL. Expression in the largely slow soleus muscle was more affected than in the EDL, and expression in the distal tongue and diaphragm muscles was affected more than in soleus. Median expression of the transgene in ventricle decreased about 18-fold upon Trex mutation. Transfections into neonatal rat myocardiocytes confirmed the importance of the Trex site for MCK enhancer activity in heart muscle, but the effect is larger in transgenic mice than in cultured cells.     

Functional Analysis of Two Matrix Attachment Region (MAR) Elements in Transgenic Maize Plants

Matrix attachment regions (MARs) are binding sites for nuclear scaffold proteins in vitro, and are proposed to mediate the attachment of chromatin to the nuclear scaffold in vivo. Previous reports suggest that MAR elements may stabilize transgene expression. Here, we tested the effects of two maize MAR elements (P-MAR from the P1-rr gene, and Adh1-MAR from the adh1 gene) on the expression of a gusA reporter gene driven by three different promoters: the maize p1 gene promoter, a wheat peroxidase (WP) gene promoter, or a synthetic promoter (Rsyn7). The inclusion of P-MAR or Adh1-MAR on P::GUS transgene constructs did not reduce variation in the levels of GUS activity among independent transformation events, nor among the progeny derived from each event. The Adh1-MAR element did not affect GUS expression driven by the WP promoter, but did modify the spatial pattern of expression of the Rsyn7::GUS transgene. These results indicate that, in transgenic maize plants, the effects of MAR elements can vary significantly depending upon the promoter used to drive the transgene.     

The Saccharomyces cerevisiae chitinase,encoded by the CTS1-2 gene,confers antifungal activity against Botrytis cinerea to Transgenic tobacco

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene has recently been confirmed by in vitro tests to possess antifungal abilities. In this study, the CTS1-2 gene has been evaluated for its in planta antifungal activity by constitutive overexpression in tobacco plants to assess its potential to increase the plant's defence against fungal pathogens. Transgenic tobacco plants, generated by Agrobacterium-mediated transformation, showed stable integration and inheritance of the transgene. Northern blot analyses conducted on the transgenic tobacco plants confirmed transgene expression. Leaf extracts from the transgenic lines inhibited Botrytis cinerea spore germination and hyphal growth by up to 70% in a quantitative in vitro assay, leading to severe physical damage on the hyphae. Several of the F₁ progeny lines were challenged with the fungal pathogen, B. cinerea, in a detached leaf infection assay, showing a decrease in susceptibility ranging from 50 to 70%. The plant lines that showed increased disease tolerance were also shown to have higher chitinase activities.     

Molecular Structure and Regulatory Potential of a T-DNA Integration Site in Petunia

The genomic structure surrounding a T-DNA integration site in a transgenic petunia plant, which shows deregulation of a root-specific promoter, was investigated. We have already demonstrated that T-DNA integration in this transformant (P13) had occurred close to a scaffold/matrix attachment region (S/MAR). A major question regarding the observed promoter leakiness was whether the T-DNA had integrated into the centre or at the border of the Petun-SAR and whether other regulatory elements are located within this genomic region. While small rearrangements were shown to occur during T-DNA integration in agreement with other reports, we find indications of the presence of a SINE retroposon – an apparent landmark for recombinogenic targets – at the integration site. Binding assays to both plant and animal nuclear scaffolds, supported by biomathematical analyses, reveal that the T-DNA is definitely located at the border of a strong S/MAR, which is in agreement with current models on the structure of integration sites. These results, together with a developmentally regulated leaf-specific enhancer effect of the Petun-SAR on gene expression in transgenic tobacco plants, indicate that the Petun-SAR demarcates the right border of a chromatin domain with genes predominantly active in leaves.     

Tritrophic Interactions Between Transgenic Potato Expressing Snowdrop Lectin (GNA), an Aphid Pest (Peach–Potato Aphid; Myzus Persicae (Sulz.) and a Beneficial Predator (2-Spot Ladybird; Adalia Bipunctata L.)

Tritrophic interactions between transgenic potato expressing the insecticidal lectin from snowdrop (Galanthus nivalis agglutinin; GNA), an aphid pest, Myzus persicae (Sulz.), and a beneficial predator, the 2-spot ladybird (Adalia bipunctata L.) were investigated. Clonal plants expressing GNA at 0.1–0.2% total soluble protein in leaves were used. No significant effects on development and survival of ladybird larvae fed on aphids from these transgenic plants were observed, with larval survival in the experimental group being 90% compared to 89% for controls. There were also no effects on subsequent female or male longevity. Female fecundity was also investigated. Although no significant differences (p > 0.05) were observed in egg production between control and experimental groups, a 10%, reduction (p < 0.01) in egg viability (determined by % hatch) occurred in ladybirds fed aphids reared on transgenic plants. Additional studies were carried out using aphids fed on artificial diet containing GNA, to deliver quantified levels of the protein to ladybird adults. GNA had no deleterious effects upon adult longevity, but resulted in a consistent trend for improved fecundity. Egg production was increased by up to 70% and egg viability also increased significantly. The results suggest that GNA is not deleterious to ladybirds. Results from these studies highlight the need to discriminate between direct and indirect effects when studying tritrophic interactions between plants/pests/natural enemies. Furthermore, it emphasises the importance of demonstrating cause and effect.     

A Sequential Approach to Risk Assessment of Transgenic Plants Expressing Protease Inhibitors: Effects on Nontarget Herbivorous Insects

Protease inhibitors expressed in transgenic plants can provide enhanced levels of resistance to important pest species. A sequential approach for testing the effects of protease inhibitor-expressing crops on nontarget herbivorous insects has been developed. The approach consists of five tiers. The first two tiers comprise the selection phase. In tier one, field surveys are used to characterise the nontarget invertebrate fauna of a crop. In tier 2, histochemical assays are used to identify the subset of herbivores with a particular class of digestive proteolytic enzymes. In the assessment phase a combination of laboratory worst-case scenario studies (tier 3) and controlled environment or small-scale field trials (tier 4) are used to evaluate the impact of the protease inhibitor-expressing plants on the selected nontarget species. In the final tier, field trials are used to compare the relative effect of transgenic plants and current management practices, such as pesticide use, on selected species. The first four tiers of the approach are described using potatoes expressing cystatins, a family of cysteine proteinase inhibitors, as an example. Although the plants have enhanced levels of resistance to potato cyst nematodes (PCN), Globodera pallida and Globodera rostochiensis, the results establish that they have negligible impact on the nontarget herbivorous insect, Eupteryx aurata.