Strategy to design and functionally analyze an miR targeted reporter gene construct using In planta assay

Plants, upon exposure to environmental stress, express sophisticated and co-ordinated responses to reprogram interconnected defense networks and metabolic pathways. These responses are governed by intricate molecular and biochemical signal transduction processes, which act in harmony to determine tolerance and sensitivity at a holistic level. Modern studies in plant stress biology include identification of key genes that influence stress tolerance and the verification of gene functions using knockout mutants or over-expression lines. Abiotic stresses induce aberrant expression of many miRNAs, suggesting that miRNAs could be a new target for genetically improving plant stress tolerance. MicroRNAs (miRNAs), 21-24 nts in length, are an extensive class of endogenous small RNA molecules that provide a fascinating option for engineering tolerance to environmental stress by serving as non-protein coding switches of both desirable and undesirable gene expressions. During abiotic stress, miRNAs function by regulatingthe stress inducing networks like signalling components and a variety of transcription factors (TFs) that leads to cleavage of the target genes. It is important to identify the expression domains of miRs and their targets to understand how the spatio-temporal regulations are co-ordinated under stress. In this project, we aim to design reporter gene construct containing the target site of selected miR. We have selected pCAMBIA1302 vector with the aim to insert the target site before the ATG of GFP gene such that the coding frame remains intact. Later, the functionality of the designed construct can be checked by expressing the constructs In planta using Agroinfiltration and the technology is expected to greatly favour in molecular pharming.

76 SCREENING Oct-4 PROMOTER ACTIVITY IN BOVINE FIRST AND SECOND ROUND SCNT EMBRYOS USING AN EGFP REPORTER CONSTRUCT

Oct-4, also known as Oct-3 or POU5F1, belongs to the POU (Pit-Oct-Unc) transcription factor family. So far it has been identified in mice, cattle, pigs, and humans. In mice, pluripotent cells of the early pre-implantation embryo express Oct-4. Therefore, Oct-4 expression is considered to be a marker for pluripotency in mice. In bovine blastocysts, Oct-4 protein is not restricted to the pluripotent cells of the inner cell mass (ICM) but is also present in the trophectoderm (TE). It has been reported, however, that at this stage the transcript level of Oct-4 was already downregulated in the TE suggesting a similar regulation of Oct-4 transcription in bovine and mice. To obtain insight into the regulation of the Oct-4 promoter after somatic cell nuclear transfer (SCNT), we transfected bovine fetal fibroblasts (BFF) with GOF18-�PE-EGFP, a reporter gene construct for the Oct-4 promoter (kindly provided by Dr. Hans R. Schoeler, Director, Max Planck Institute for Molecular Biomedicine, M�nster, Germany). Six stably transfected colonies (BFFGOF), none of which exhibited green fluorescence, were used for SCNT, with subsequent examination of the resulting embryos on Days 5-7 by fluorescence microscopy. SCNT embryos originating from BFF colonies 3, 12, and 82 showed fluorescence both in ICM and TE. SCNT with BFF colony 8 resulted in embryos with no detectable fluorescence. This might have been due to a positional effect of the reporter gene construct or an incomplete reprogramming of the used fibroblast colonies during SCNT. To study the consequences of another round of SCNT on Oct-4 promoter activity, cloned embryos from BFF colonies 12 (positive) and 8 (negative) were transferred to recipients. At Day 33, three BFFGOF12 and four BFFGOF8 fetuses were recovered and BFF cultures were established from all cloned fetuses. The presence of GOF18-�PE-EGFP was verified by PCR and Southern blot analysis. BFF cultures from two cloned BFFGOF12 and BFFGOF8 fetuses each were used for SCNT, and fluorescence was examined in Day 6 embryos. SCNT embryos derived from BFFGOF12 fetuses exhibited weaker fluorescence than embryos directly derived from the original transfected colony. SCNT embryos derived from BFFGOF8 fetuses showed no fluorescence. GFP-positive embryos will be examined by immunocytochemistry using an antibody against Oct-4 to evaluate the correlation between endogenous Oct-4 and Oct-4 reporter gene expression. Further fluorescence in situ hybridization (FISH) analyses are underway to localize the reporter gene integration sites in BFFGOF8 and BFFGOF12. In summary, stable GOF18-�PE-EGFP transfected cells are an interesting tool for monitoring Oct-4 promoter activation after using different protocols of SCNT or in consecutive rounds of SCNT. Furthermore, it will be possible to correlate Oct-4 promoter activity with epigenetic mechanisms, such as DNA methylation and histone modifications, in SCNT embryos.

Construction and Preliminary Evaluation of an Aspergillus flavus Reporter Gene Construct as a Potential Tool for Screening Aflatoxin Resistance

Effective preharvest strategies to eliminate aflatoxin accumulation in crops are not presently available. The molecular biology of aflatoxin biosynthesis has been extensively studied, and genetic and molecular tools such as reporter gene systems for the measurement of fungal growth have been developed. A reporter construct containing the Aspergillus flavus β-tubulin gene promoter fused to Escherichia coli β-glucuronidase (GUS) has been shown to be a reliable tool for the indirect measurement of fungal growth in maize kernels. Since cost-saving alternative methods for the direct measurement of aflatoxin levels are needed to facilitate more widespread field and laboratory screening of maize lines, a new reporter gene construct involving the promoter region of the omtA gene of the aflatoxin biosynthetic pathway was constructed and tested. Expression of GUS activity by this construct (omtA::GUS) was correlated with aflatoxin accumulation in culture. In the fungal transformant GAP26-1, which harbors this construct, aflatoxin production and GUS expression on sucrose-containing medium showed the same temporal pattern of toxin induction. Furthermore, GUS expression by GAP26-1 was shown to be associated with aflatoxin accumulation in maize kernels inoculated with this strain. Our results suggest that this and other reporter gene pathway promoter constructs may provide superior alternatives to direct aflatoxin quantification with respect to time, labor, and materials for the screening of maize lines for resistance to aflatoxin accumulation.

A type I collagen reporter gene construct for protein engineering studies. Functional equivalence of transfected reporter COL1A1 and endogenous gene products during biosynthesis and in vitro extracellular matrix accumulation

A type I collagen reporter gene construct, designed to facilitate detailed analysis of the consequences of introduced structural and regulatory mutations on collagen biosynthesis and participation in the extracellular matrix, was produced by site-directed mutagenesis of the mouse COL1A1 gene. The reporter construct, pWTCI-Ile822, carried a single base change which converted the codon for amino acid 822 of the triple helix from methionine to isoleucine. This change allowed the reporter protein, [Ile822]alpha 1(I), to be distinguished from the wild-type alpha 1(I), and quantified, by its altered CNBr cleavage pattern. In mouse Mov13 cells, which synthesize no endogenous pro alpha 1(I), reporter chains associated with endogenous pro alpha 2(I), formed pepsin-stable triple helices and were secreted efficiently from the cell. The thermal stability of wild-type molecules and molecules containing the reporter [Ile822]alpha 1(I) chains was identical. The biosynthetic characteristics of wild-type and reporter chains were directly compared in stably transfected 3T6 cells. These cells did not make a distinction between reporter and endogenous alpha 1(I) chains, which were secreted from the cells at the same rate and were processed and deposited into the 3T6 cell in vitro accumulated extracellular matrix with equal efficiency. These data demonstrate that the helical sequence alteration in the reporter protein is functionally neutral and that the reporter construct, pWTCI-Ile822, is a suitable vector for the analysis of the biochemical effects of site-directed mutations in the putative COL1A1 functional domains.