Phage Bxb1 integrase mediates highly efficient site-specific recombination in mammalian cells.
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Biomedical subjects
Malla Padidam
Publications and source records attributed to Malla Padidam.
Inducible control of transgene expression with ecdysone receptor: gene switches with high sensitivity, robust expression, and reduced size.
The ecdysone receptor (EcR)-based gene regulation system is a tool for controlling gene expression. To improve the sensitivity of this system, we evaluated many two-hybrid format synthetic gene constructs in which the GAL4 DNA binding domain was fused to the ligand binding domain of the Choristoneura fumiferana EcR mutant V390I/Y410E (GEvy), and various activation domains--VP16, p53, p65, or E2F-i--were fused to the EF domains of chimeric human RXR. These gene switches were assayed in NIH3T3 cells, HEK293 cells, and in mouse quadriceps in the presence of the nonsteroidal inducer RG-115819 or GS-E. All of the two-hybrid format constructs had no or very low background in the "off" condition and high luciferase reporter gene expression levels in "on" conditions. Extremely high sensitivity was achieved, with EC50 values in the subnanomolar range and with maximal induction at 10 nM RG-115819. Co-expression of both receptor genes with encephalomyocarditis virus (EMCV) or eIF4G internal ribosome entry site (IRES) sequences gave robust induction levels. To reduce the size of the switch construct, we tested single receptor formats, in which any of 14 different activation domains were fused to GEvy. We identified several switches with acceptable levels of basal and maximal induction levels. The gene switches described here provide receptor configuration options suitable for gene function studies, therapeutic protein production in cell culture, transgenic mouse models, and gene/cell therapy.
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Chemically regulated gene expression in plants.
Chemically inducible systems that activate or inactivate gene expression have many potential applications in the determination of gene function and in plant biotechnology. The precise timing and control of gene expression are important aspects of chemically inducible systems. Several systems have been developed and used to analyze gene function, marker-free plant transformation, site-specific DNA excision, activation tagging, conditional genetic complementation, and restoration of male fertility. Chemicals that are used to regulate transgene expression include the antibiotic tetracycline, the steroids dexamethasone and estradiol, copper, ethanol, the inducer of pathogen-related proteins benzothiadiazol, herbicide safeners, and the insecticide methoxyfenozide. Systems that are suitable for field application are particularly useful for experimental systems and have potential applications in biotechnology.
Copper↗
Chemical-inducible, ecdysone receptor-based gene expression system for plants.
We have developed an inducible gene expression system with potential for field application using the ecdysone receptor (EcR) from the spruce budworm and the non-steroidal EcR agonist, methoxyfenozide. Chimeric transcription activators were constructed with EcR ligand binding domain, GAL4 and LexA DNA binding domains, and VP16 activation domain. In the presence of methoxyfenozide, the transcription activators induced expression of the luciferase reporter gene cloned downstream of a promoter containing GAL4A- or LexA-response element and a minimal 35S promoter. Low basal and high induced luciferase expression was optimized by cloning the activator and the reporter genes in different tandem orientations. Many transgenic Arabidopsis and tobacco plants were obtained with little or no basal expression in the absence of methoxyfenozide and inducible expression that was several fold higher than that observed with the constitutive 35S promoter. Moreover, gene expression was controlled over a wide range of methoxyfenozide concentration. Our results demonstrate that the inducible gene expression system based on the spruce budworm EcR ligand binding domain with methoxyfenozide as a ligand is very effective in regulating transgenes in plants. It is suitable for field applications because methoxyfenozide is commercially available and has an exceptional health and environmental safety profile.
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