Agrobacterium rhizogenes transformation of the Phaseolus spp.: A tool for functional genomics

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: R coccineus, P lunatus, and P acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions.

NADH-Glutamate Synthase in Alfalfa Root Nodules. Genetic Regulation and Cellular Expression1

NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen assimilation in alfalfa (Medicago sativa L.) root nodules. Here we report that in alfalfa, a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type cv Saranac nodules the NADH-GOGAT gene is predominantly expressed in infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone was observed. In 33-d-old nodules expression was seen in only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric β-glucuronidase promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88-bp direct repeat.

Studying Early Nodulin Gene ENOD40 Expression and Induction by Nodulation Factor and Cytokinin in Transgenic Alfalfa1

ENOD40, an early nodulin gene, is expressed following inoculation with Rhizobium meliloti or by adding R. meliloti-produced nodulation (Nod) factors or the plant hormone cytokinin to uninoculated roots. We isolated two MsENOD40 clones, designated MsENOD40–1 and MsENOD40–2, with distinct promoters from an alfalfa (Medicago sativa cv Chief) genomic library. The promoters were fused to the reporter gene uidA (gus), and the constructs were introduced into alfalfa. We observed that the MsENOD40–1 construct was expressed almost exclusively under symbiotic conditions. The MsENOD40–2 construct was transcribed under both symbiotic and nonsymbiotic conditions and in nonnodular and nodular tissues. Both MsENOD40 promoter-gus constructs were similarly expressed as nodules developed, and both were expressed in roots treated with 6-benzylaminopurine or purified Nod factor. However, no blue color was detected in nodule-like structures induced by the auxin transport inhibitor N-1-(naphthyl)phthalamic acid on roots of plants containing the MsENOD40–1 promoter construct, whereas pseudonodules from plants containing the MsENOD40–2 promoter construct stained blue. A 616-bp region at the distal 5′ end of the promoter is important for proper spatial expression of MsENOD40 in nodules and also for Nod-factor and cytokinin-induced expression.

Antiangiogenic therapy of transgenic mice impairs de novo tumor growth.

Angiogenesis is activated during multistage tumorigenesis prior to the emergence of solid tumors. Using a transgenic mouse model, we have tested the proposition that treatment with angiogenesis inhibitors can inhibit the progression of tumorigenesis after the switch to the angiogenic phenotype. In this model, islet cell carcinomas develop from multifocal, hyperproliferative nodules that show the histological hallmarks of human carcinoma in situ. Mice were treated with a combination of the angiogenesis inhibitor AGM-1470 (TNP-470), the antibiotic minocycline, and interferon alpha/beta. The treatment regimen markedly attenuated tumor growth but did not prevent tumor formation; tumor volume was reduced to 11% and capillary density to 40% of controls. The proliferation index of tumor cells in treated and control mice was similar, whereas the apoptotic index was doubled in treated tumors. This study shows that de novo tumor progression can be restricted solely by antiangiogenic therapy. The results suggest that angiogenesis inhibitors represent a valid component of anticancer strategies aimed at progression from discrete stages of tumorigenesis and demonstrate that transgenic mouse models can be used to evaluate efficacy of candidate antiangiogenic agents.

Strategies to obtain stable transgenic plants from non-embryogenic lines: complementation of the nn(1) mutation of the NORK gene in Medicago sativa MN1008

Strategies to introduce genes into non-embryogenic plants for complementation of a mutation are described and tested on tetraploid alfalfa (Medicago sativa). Genes conditioning embryogenic potential, a mutant phenotype, and a gene to complement the mutation can be combined using several different crossing and selection steps. In the successful strategy used here, the M. sativa genotype MnNC-1008(NN) carrying the recessive non-nodulating mutant allele nn(1) was crossed with the highly embryogenic alfalfa line Regen S and embryogenic hybrid individuals were identified from the F1 progeny. After transformation of these hybrids with the wild-type gene (NORK), an F2 generation segregating for the mutation and transgene were produced. Plants homozygous for the mutant allele and carrying the wild-type NORK transgene could form root nodules after inoculation with Sinorhizobium meliloti demonstrating successful complementation of the nn(1) mutation.

Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane

A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.

Modeling prostate cancer: a perspective on transgenic mouse models

Despite considerable success in treatment of early stage localized prostate cancer (PC), acute inadequacy of late stage PC treatment and its inherent heterogeneity poses a formidable challenge. Clearly, an improved understanding of PC genesis and progression along with the development of new targeted therapies are warranted. Animal models, especially, transgenic immunocompetent mouse models, have proven to be the best ally in this respect. A series of models have been developed by modulation of expression of genes implicated in cancer-genesis and progression; mainly, modulation of expression of oncogenes, steroid hormone receptors, growth factors and their receptors, cell cycle and apoptosis regulators, and tumor suppressor genes have been used. Such models have contributed significantly to our understanding of the molecular and pathological aspects of PC initiation and progression. In particular, the transgenic mouse models based on multiple genetic alterations can more accurately address the inherent complexity of PC, not only in revealing the mechanisms of tumorigenesis and progression but also for clinically relevant evaluation of new therapies. Further, with advances in conditional knockout technologies, otherwise embryonically lethal gene changes can be incorporated leading to the development of new generation transgenics, thus adding significantly to our existing knowledge base. Different models and their relevance to PC research are discussed.

Broadening of transgenic adenocarcinoma of the mouse prostate (TRAMP) model to represent late stage androgen depletion independent cancer

BACKGROUND The transgenic adenocarcinoma of the mouse prostate (TRAMP) model closely mimics PC-progression as it occurs in humans. However, the timing of disease incidence and progression (especially late stage) makes it logistically difficult to conduct experiments synchronously and economically. The development and characterization of androgen depletion independent (ADI) TRAMP sublines are reported. METHODS Sublines were derived from androgen-sensitive TRAMP-C1 and TRAMP-C2 cell lines by androgen deprivation in vitro and in vivo. Epithelial origin (cytokeratin) and expression of late stage biomarkers (E-cadherin and KAI-1) were evaluated using immunohistochemistry. Androgen receptor (AR) status was assessed through quantitative real time PCR, Western blotting, and immunohistochemistry. Coexpression of AR and E-cadherin was also evaluated. Clonogenicity and invasive potential were measured by soft agar and matrigel invasion assays. Proliferation/survival of sublines in response to androgen was assessed by WST-1 assay. In vivo growth of subcutaneous tumors was assessed in castrated and sham-castrated C57BL/6 mice. RESULTS The sublines were epithelial and displayed ADI in vitro and in vivo. Compared to the parental lines, these showed (1) significantly faster growth rates in vitro and in vivo independent of androgen depletion, (2) greater tumorigenic, and invasive potential in vitro. All showed substantial downregulation in expression levels of tumor suppressor, E-cadherin, and metastatis suppressor, KAI-1. Interestingly, the percentage of cells expressing AR with downregulated E-cadherin was higher in ADI cells, suggesting a possible interaction between the two pathways. CONCLUSIONS The TRAMP model now encompasses ADI sublines potentially representing different phenotypes with increased tumorigenicity and invasiveness.