A resource for the conditional ablation of microRNAs in the mouse

The importance of miRNAs during development and disease processes is well established. However, most studies have been done in cells or with patient tissues, and therefore the physiological roles of miRNAs are not well understood. To unravel in vivo functions of miRNAs, we have generated conditional, reporter-tagged knockout-first mice for numerous evolutionarily conserved miRNAs. Here, we report the generation of 162 miRNA targeting vectors, 64 targeted ES cell lines, and 46 germline-transmitted miRNA knockout mice. In vivo lacZ reporter analysis in 18 lines revealed highly tissue-specific expression patterns and their miRNA expression profiling matched closely with published expression data. Most miRNA knockout mice tested were viable, supporting a mechanism by which miRNAs act redundantly with other miRNAs or other pathways. These data and collection of resources will be of value for the in vivo dissection of miRNA functions in mouse models.     

A genome-scale shRNA resource for transgenic RNAi in Drosophila

Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.     

A densely interconnected genome-wide network of microRNAs and oncogenic pathways revealed using gene expression signatures

MicroRNAs (miRNAs) are important components of cellular signaling pathways, acting either as pathway regulators or pathway targets. Currently, only a limited number of miRNAs have been functionally linked to specific signaling pathways. Here, we explored if gene expression signatures could be used to represent miRNA activities and integrated with genomic signatures of oncogenic pathway activity to identify connections between miRNAs and oncogenic pathways on a high-throughput, genome-wide scale. Mapping >300 gene expression signatures to >700 primary tumor profiles, we constructed a genome-wide miRNA-pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA-pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Globally, the miRNA-pathway network demonstrates a small-world, but not scale-free, organization characterized by multiple distinct, tightly knit modules each exhibiting a high density of connections. However, unlike genetic or metabolic networks typified by only a few highly connected nodes ("hubs"), most nodes in the miRNA-pathway network are highly connected. Sequence-based computational analysis confirmed that highly-interconnected miRNAs are likely to be regulated by common pathways to target similar sets of downstream genes, suggesting a pervasive and high level of functional redundancy among coexpressed miRNAs. We conclude that gene expression signatures can be used as surrogates of miRNA activity. Our strategy facilitates the task of discovering novel miRNA-pathway connections, since gene expression data for multiple normal and disease conditions are abundantly available.     

A one-plasmid conditional color-switching transgenic system for multimodal bioimaging

We have developed a new construct to generate transgenic mice with one plasmid that offers: (1) Cre/loxP-mediated spatial and temporally-controlled tissue-specific transgene expression; (2) A color-switching mechanism that uses spectrum-complementary genetically-encoded red (mRFP) and green (eGFP) fluorescent markers to label the transgene-expressing cells; (3) A bioluminescent marker that turns-on in the transgene-expressing cells; (4) eGFP as a cell surface marker in the transgene-expressing cells that facilitates the isolation and targeting of these cells. This vector was tested in vitro by co-transfection of the transgenic plasmid and a plasmid containing Cre recombinase into cultured cells and by establishing a transgenic mouse line. We show that this method allows versatile transgene expression targeting and color-switching to facilitate fluorescent and bioluminescent imaging both in cultured cells and in vivo. Our strategy provides time-saving features in tissue-specific transgene expression, bioimaging and primary cell isolation and can be used for generation of gene-specific transgenic mice.     

A genome-wide search for synaptic vesicle cycle proteins in Drosophila

The recent completion of the Drosophila genome sequence opens new avenues for neurobiology research. We screened the fly genome sequence for homologs of mammalian genes implicated directly or indirectly in exocytosis and endocytosis of synaptic vesicles. We identified fly homologs for 93% of the vertebrate genes that were screened. These are on average 60% identical and 74% similar to their vertebrate counterparts. This high degree of conservation suggests that little protein diversification has been tolerated in the evolution of synaptic transmission. Finally, and perhaps most exciting for Drosophila neurobiologists, the genomic sequence allows us to identify P element transposon insertions in or near genes, thereby allowing rapid isolation of mutations in genes of interest. Analysis of the phenotypes of these mutants should accelerate our understanding of the role of numerous proteins implicated in synaptic transmission.     

Targeted expression of a conditional oncogene in hematopoietic cells of transgenic mice

We have produced two lines of transgenic mice in which the expression of temperature-sensitive SV-40 large T antigen is targeted to bone marrow megakaryocytes via the platelet factor 4 (PF4) tissue-specific promoter. The progeny of these transgenic mice were observed for about 3 mo, and no malignancies were detected over this period of time. The offspring of these transgenic mice, 6- to 12-wk of age, served as a source of bone marrow cells, which upon in vitro cultivation at the permissive temperature yielded immortalized cell lines (MegT). At the permissive temperature, MegT cells exhibit the characteristics of early 2N and 4N megakaryocytes which include the presence of specific gene products such as PF4, glycoprotein IIb, acetylcholinesterase, and CD45 as well as the absence of molecular markers of other cell lineages such as the macrophage marker Mac-1, the T helper cell marker CD4, the mast cell marker IgE, the T cell marker CD2 or the erythroid cell marker alpha-globin. The inactivation of the oncogene by a shift of temperature from 34 degrees to 39.5 degrees C produces a reduction in the frequency of the 2N cells, in conjunction with the appearance of 8N and 16N cells, consisting of 27 and 3% of total cells, respectively. Thus, we have generated hematopoietic cell lines that are trapped in the early stages of megakaryocyte commitment, but able to undergo part of the normal program of terminal differentiation.     

Design and utility of temperature-sensitive Gal4 mutants for conditional gene expression in Drosophila

Temperature-sensitive (ts) mutants are valuable tools to study the function of essential genes in vivo. Despite their widespread use, little is known about mechanisms responsible for the temperature-sensitive (ts) phenotype, or of the transferability of ts mutants of a specific gene between organisms. Since ts mutants are typically generated by random mutagenesis it is difficult to isolate such mutants without efficient screening procedures. We have recently shown that it is possible to obtain ts mutants at high frequency by targeted mutations at either predicted, buried residues important for protein stability or at functional, ligand binding residues. The former class of residues can be identified solely from amino acid sequence and the latter from Ala scanning mutagenesis or from a structure of the protein:ligand complex. Several ts mutants of Gal4 in yeast were generated by mutating both categories of residues. Two of these ts mutants were also shown to result in tight and rapid ts reporter gene-expression in Drosophila when driven by either the elav or GMR promoters. We suggest possible mechanisms that might be responsible for such transferable ts phenotypes and also discuss some of the limitations and difficulties involved in rational design of ts mutants.     

Human cytomegalovirus immediate-early-gene expression disrupts embryogenesis in transgenic Drosophila

Intrauterine infection with human cytomegalovirus (HCMV) is the leading viral cause of birth defects involving the central nervous system. Due to the highly species specific nature of the virus, its course of natural infection cannot be studied in animal models. Here we introduce a novel transgenic Drosophila model system for studying the effects of the major viral regulatory genes, the immediate-early genes, on normal embryonic development. We show that ectopic expression of the immediate-early genes in Drosophila led to increased embryonic lethality manifested in disintegration of the embryos. Further analysis suggested that immediate-early gene expression interfered with adherens junction maintenance, leading to the disruption of embryonic epithelial integrity. Owing to the evolutionary conservation of developmental mechanisms from invertebrates to mammals, we anticipate that the studies in Drosophila will be relevant also to humans and will ultimately provide a versatile system for studying different aspects of viral-host interactions.     

A genome-wide, end-sequenced 129Sv BAC library resource for targeting vector construction

The majority of gene-targeting experiments in mice are performed in 129Sv-derived embryonic stem (ES) cell lines, which are generally considered to be more reliable at colonizing the germ line than ES cells derived from other strains. Gene targeting is reliant on homologous recombination of a targeting vector with the host ES cell genome. The efficiency of recombination is affected by many factors, including the isogenicity (H. te Riele et al., 1992, Proc. Natl. Acad. Sci. USA 89, 5128-5132) and the length of homologous sequence of the targeting vector and the location of the target locus. Here we describe the double-end sequencing and mapping of 84,507 bacterial artificial chromosomes (BACs) generated from AB2.2 ES cell DNA (129S7/SvEvBrd-Hprtb-m2). We have aligned these BACs against the mouse genome and displayed them on the Ensembl genome browser, DAS: 129S7/AB2.2. This library has an average insert size of 110.68 kb and average depth of genome coverage of 3.63- and 1.24-fold across the autosomes and sex chromosomes, respectively. Over 97% of the mouse genome and 99.1% of Ensembl genes are covered by clones from this library. This publicly available BAC resource can be used for the rapid construction of targeting vectors via recombineering. Furthermore, we show that targeting vectors containing DNA recombineered from this BAC library can be used to target genes efficiently in several 129-derived ES cell lines.     

A resource of vectors and ES cells for targeted deletion of microRNAs in mice

The 21-23 nucleotide, single-stranded RNAs classified as microRNAs (miRNA) perform fundamental roles in diverse cellular and developmental processes. In contrast to the situation for protein-coding genes, no public resource of miRNA mouse mutant alleles exists. Here we describe a collection of 428 miRNA targeting vectors covering 476 of the miRNA genes annotated in the miRBase registry. Using these vectors, we generated a library of highly germline-transmissible C57BL/6N mouse embryonic stem (ES) cell clones harboring targeted deletions for 392 miRNA genes. For most of these targeted clones, chimerism and germline transmission can be scored through a coat color marker. The targeted alleles have been designed to be adaptable research tools that can be efficiently altered by recombinase-mediated cassette exchange to create reporter, conditional and other allelic variants. This miRNA knockout (mirKO) resource can be searched electronically and is available from ES cell repositories for distribution to the scientific community.     

A construct with fluorescent indicators for conditional expression of miRNA

Background  

Transgenic RNAi holds promise as a simple, low-cost, and fast method for reverse genetics in mammals. It may be particularly useful for producing animal models for hypomorphic gene function. Inducible RNAi that permits spatially and temporally controllable gene silencing in vivo will enhance the power of transgenic RNAi approach. Furthermore, because microRNA (miRNA) targeting specific genes can be expressed simultaneously with protein coding genes, incorporation of fluorescent marker proteins can simplify the screening and analysis of transgenic RNAi animals.     

Bioavailability of transgenic microRNAs in genetically modified plants

Background

Transgenic expression of small RNAs is a prevalent approach in agrobiotechnology for the global enhancement of plant foods. Meanwhile, emerging studies have, on the one hand, emphasized the potential of transgenic microRNAs (miRNAs) as novel dietary therapeutics and, on the other, suggested potential food safety issues if harmful miRNAs are absorbed and bioactive. For these reasons, it is necessary to evaluate the bioavailability of transgenic miRNAs in genetically modified crops.

Results

As a pilot study, two transgenic Arabidopsis lines ectopically expressing unique miRNAs were compared and contrasted with the plant bioavailable small RNA MIR2911 for digestive stability and serum bioavailability. The expression levels of these transgenic miRNAs in Arabidopsis were found to be comparable to that of MIR2911 in fresh tissues. Assays of digestive stability in vitro and in vivo suggested the transgenic miRNAs and MIR2911 had comparable resistance to degradation. Healthy mice consuming diets rich in Arabidopsis lines expressing these miRNAs displayed MIR2911 in the bloodstream but no detectable levels of the transgenic miRNAs.

Conclusions

These preliminary results imply digestive stability and high expression levels of miRNAs in plants do not readily equate to bioavailability. This initial work suggests novel engineering strategies be employed to enhance miRNA bioavailability when attempting to use transgenic foods as a delivery platform.