Optimization of Cell-Based cDNA Microarray Conditions for Gene Functional Studies in HEK293 Cells.

Since the cell-based cDNA microarray (CBCM) technique has been a useful tool for gain-of-function studies, many investigators have used CBCMs to identify interesting genes. However, this method requires better-established conditions to ensure high reverse transfection efficiency without cross-contamination. Therefore, we optimized CBCM techniques through various means. We determined that Lipofectamine 2000 was the most appropriate transfection reagent by evaluating eight commercialized reagents, and we determined that the most effective concentrations for printing solution constituents were 0.2 M sucrose (to yield a final concentration of 32 mM) and 0.2% gelatin (to yield a final concentration 0.075%). After examining various combinations, we also determined that the best concentrations of cDNA and transfection reagent for optimal reverse transfection efficiency were 1.5 µg/5 µL of cDNA and 5.5 µL of Lipofectamine 2000. Finally, via a time course, we determined that 72 h was the most effective reaction duration for reverse transfection, and we confirmed the stability of cDNA spot activity of CBCMs for various storage periods. In summary, the CBCM conditions that we have identified can provide more effective outcomes for cDNA reverse transfection on microarrays.

Development of Cell-Defined Lentivirus-Based Microarray for Mammalian Cells.

Although reverse transfection cell microarray (RTCM) is a powerful tool for mammalian cell studies, the technique is not appropriate for cells that are difficult to transfect. The lentivirus-infected cell microarray (LICM) technique was designed to improve overall efficiency. However, LICM presents new challenges because individual lentiviral particles can spread through the cell population, leading to cross-contamination. Therefore, we designed a cell-defined lentivirus microarray (CDLM) technique using cell-friendly biomaterials that are controlled by cell attachment timing. We selected poly-l-lysine (PLL) with Matrigel as the best combination of biomaterials for cell-defined culture. We used 2 µL PLL to determine by titration the optimum concentration required (0.04% stock, 0.005% final concentration). We also determined the optimum concentration of 10 µL of lentivirus particles for maximum reverse infection efficiency (1 × 108 infectious units [IFU]/mL stock, 62.5% final concentration) and established the best combination of components for the lentivirus mixture (10 µL of lentivirus particles and 2 µL each of siGLO Red dye, Matrigel, and 0.04% PLL). Finally, we validated both the effect of reverse infection in various cell lines and lentivirus spot activity in CDLM by storage period. This method provides an effective lentivirus-infected cell microarray for large-scale gene function studies.

The monitoring of gene functions on a cell-defined siRNA microarray in human bone marrow stromal and U2OS cells.

Here, we developed a cell defined siRNA microarray (CDSM) for human bone marrow stromal cells (hBMSCs) designed to control the culture of cells inside the spot area without reducing the efficiency of siRNA silencing, "Development of a cell-defined siRNA microarray for analysis of gene functionin human bone marrow stromal cells" (Kim et al., 2016 [1]). First, we confirmed that p65 protein inhibition efficiency was maintained when hBMSCs were culture for 7 days on the siRNA spot, and siRNA spot activity remained in spite of long term storage (10 days and 2 months). Additionally, we confirmed p65 protein inhibition in U2OS cells after 48 h reverse transfection.

Development of a cell-defined siRNA microarray for analysis of gene function in human bone marrow stromal cells.

Small interfering RNA (siRNA) screening approaches have provided useful tools for the validation of genetic functions; however, image-based siRNA screening using multiwell plates requires large numbers of cells and time, which could be the barrier in application for gene mechanisms study using human adult cells. Therefore, we developed the advanced method with the cell-defined siRNA microarray (CDSM), for functional analysis of genes in small scale within slide glass using human bone marrow stromal cells (hBMSCs). We designed cell spot system with biomaterials (sucrose, gelatin, poly-L-lysine and matrigel) to control the attachment of hBMSCs inside spot area on three-dimensional (3D) hydrogel-coated slides. The p65 expression was used as a validation standard which described our previous report. For the optimization of siRNA mixture, first, we detected five kinds of commercialized reagent (Lipofectamine 2000, RNAi-Max, Metafectine, Metafectine Pro, TurboFectin 8.0) via validation. Then, according to quantification of p65 expression, we selected 2 µl of RNAi-Max as the most effective reagent condition on our system. Using same validation standard, we optimized sucrose and gelatin concentration (80 mM and 0.13%), respectively. Next, we performed titration of siRNA quantity (2.66-5.55 µM) by reverse transfection time (24 h, 48 h, 72 h) and confirmed 3.75 µM siRNA concentration and 48 h as the best condition. To sum up the process for optimized CDSM, 3 µl of 20 µM siRNA (3.75 µM) was transferred to the 384-well V-bottom plate containing 2 µl of dH2O and 2 µl of 0.6M sucrose (80 mM). Then, 2 µl of RNAi-Max was added and incubated for 20 min at room temperature after mixing gently and centrifugation shortly. Five microliters of gelatin (0.26%) and 2 µl of growth factor reduced phenol red-free matrigel (12.5%) were added and mixed by pipetting gently. Finally, optimized siRNA mixture was printed on 3D hydrogel-coated slides and cell-defined attachment and siRNA reverse transfection were induced. The efficiency of this CDSM was verified using three siRNAs (targeting p65, Slug, and N-cadherin), with persistent gene silencing for 5 days. We obtained the significant and reliable data with effective knock-down in our condition, and suggested our method as the qualitatively improved siRNA microarray screening method for hBMSCs.

Phenotypic MicroRNA Microarrays.

Microarray technology has become a very popular approach in cases where multiple experiments need to be conducted repeatedly or done with a variety of samples. In our lab, we are applying our high density spots microarray approach to microscopy visualization of the effects of transiently introduced siRNA or cDNA on cellular morphology or phenotype. In this publication, we are discussing the possibility of using this micro-scale high throughput process to study the role of microRNAs in the biology of selected cellular models. After reverse-transfection of microRNAs and siRNA, the cellular phenotype generated by microRNAs regulated NF-κB expression comparably to the siRNA. The ability to print microRNA molecules for reverse transfection into cells is opening up the wide horizon for the phenotypic high content screening of microRNA libraries using cellular disease models.

Quantum dot-based screening system for discovery of g protein-coupled receptor agonists.

Cellular imaging has emerged as an important tool to unravel biological complexity and to accelerate the drug-discovery process, including cell-based screening, target identification, and mechanism of action studies. Recently, semiconductor nanoparticles known as quantum dots (QDs) have attracted great interest in cellular imaging applications due to their unique photophysical properties such as size, tunable optical property, multiplexing capability, and photostability. Herein, we show that QDs can also be applied to assay development and eventually to high-throughput/content screening (HTS/HCS) for drug discovery. We have synthesized QDs modified with PEG and primary antibodies to be used as fluorescent probes for a cell-based HTS system. The G protein-coupled receptor (GPCR) family is known to be involved in most major diseases. We therefore constructed human osteosarcoma (U2OS) cells that specifically overexpress two types of differently tagged GPCRs: influenza hemagglutinin (HA) peptide-tagged κ-opioid receptors (κ-ORs) and GFP-tagged A3 adenosine receptors (A3AR). In this study, we have demonstrated that 1) anti-HA antibody-conjugated QDs could specifically label HA-tagged κ-ORs, 2) subsequent treatment of QD-tagged GPCR agonists allowed agonist-induced translocation to be monitored in real time, 3) excellent emission spectral properties of QD permitted the simultaneous detection of two GPCRs in one cell, and 4) the robust imaging capabilities of the QD-antibody conjugates could lead to reproducible quantitative data from high-content cellular images. These results suggest that the present QD-based GPCR inhibitor screening system can be a promising platform for further drug screening applications.

Automated genome-wide visual profiling of cellular proteins involved in HIV infection.

Recent genome-wide RNAi screens have identified >842 human genes that affect the human immunodeficiency virus (HIV) cycle. The list of genes implicated in infection differs between screens, and there is minimal overlap. A reason for this variance is the interdependence of HIV infection and host cell function, producing a multitude of indirect or pleiotropic cellular effects affecting the viral infection during RNAi screening. To overcome this, the authors devised a 15-dimensional phenotypic profile to define the viral infection block induced by CD4 silencing in HeLa cells. They demonstrate that this phenotypic profile excludes nonspecific, RNAi-based side effects and viral replication defects mediated by silencing of housekeeping genes. To achieve statistical robustness, the authors used automatically annotated RNAi arrays for seven independent genome-wide RNAi screens. This identified 56 host genes, which reliably reproduced CD4-like phenotypes upon HIV infection. The factors include 11 known HIV interactors and 45 factors previously not associated with HIV infection. As proof of concept, the authors confirmed that silencing of PAK1, Ku70, and RNAseH2A impaired HIV replication in Jurkat cells. In summary, multidimensional, visual profiling can identify genes required for HIV infection.

Visual genome-wide RNAi screening to identify human host factors required for Trypanosoma cruzi infection.

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy.

Alternative promotion of the mouse acyl-CoA synthetase 6 (mAcsl6) gene mediates the expression of multiple transcripts with 5'-end heterogeneity: genetic organization of mAcsl6 variants.

We report four variants and alternative promoter usage for the mouse acyl-CoA synthetase 6 (mAcsl6) gene. The variants, which were organized into 26 exons and 25 introns spanning 55 kb of DNA on mouse chromosome 11, were classified according to their 5'-UTRs and alternative splicing of exon 13. Alignment of the nucleotide sequences showed that the mAcsl6 variant 1 (mAcsl6_v1) and mAcsl6_v2 used a different promoter and had different splicing patterns than mAcsl6_v3 and mAcsl6_v4. The results of the promoter analysis suggest that the mAcsl6 promoter 1 (mAcsl6_pr1) region has a negative regulatory function. To verify this result, we constructed id vector constructs that contained the promoter regions mAcsl6_pr1 and 2, and the chimeric transcript. Although the mAcsl6_pr1 region was deleted, the mAcsl6_v1 and 2 transcripts were detected consistently.