Bioluminescence Imaging for Assessment and Normalization in Transfected Cell Arrays

Transfected cell arrays (TCAs) represent a high-throughput technique to correlate gene expression with functional cell responses. Despite advances in TCAs, improvements are needed for the widespread application of this technology. We have developed a TCA that combines a two-plasmid system and dual-bioluminescence imaging to quantitatively normalize for variability in transfection and increase sensitivity. The two-plasmids consist of: (i) normalization plasmid present within each spot, and (ii) functional plasmid that varies between spots, responsible for the functional endpoint of the array. Bioluminescence imaging of dual-luciferase reporters (renilla, firefly luciferase) provides sensitive and quantitative detection of cellular response, with minimal post-transfection processing. The array was applied to quantify estrogen receptor α (ERα) activity in MCF-7 breast cancer cells. A plasmid containing an ERα-regulated promoter directing firefly luciferase expression was mixed with a normalization plasmid, complexed with cationic lipids and deposited into an array. ER induction mimicked results obtained through traditional assays methods, with estrogen inducing luciferase expression 10-fold over the antiestrogen fulvestrant or vehicle. Furthermore, the array captured a dose response to estrogen, demonstrating the sensitivity of bioluminescence quantification. This system provides a tool for basic science research, with potential application for the development of patient specific therapies

Bioluminescence Imaging for Assessment and Normalization in Transfected Cell Arrays

Transfected cell arrays (TCAs) represent a high-throughput technique to correlate gene expression with functional cell responses. Despite advances in TCAs, improvements are needed for the widespread application of this technology. We have developed a TCA that combines a two-plasmid system and dual-bioluminescence imaging to quantitatively normalize for variability in transfection and increase sensitivity. The two-plasmids consist of: (i) normalization plasmid present within each spot, and (ii) functional plasmid that varies between spots, responsible for the functional endpoint of the array. Bioluminescence imaging of dual-luciferase reporters (renilla, firefly luciferase) provides sensitive and quantitative detection of cellular response, with minimal post-transfection processing. The array was applied to quantify estrogen receptor α (ERα) activity in MCF-7 breast cancer cells. A plasmid containing an ERα-regulated promoter directing firefly luciferase expression was mixed with a normalization plasmid, complexed with cationic lipids and deposited into an array. ER induction mimicked results obtained through traditional assays methods, with estrogen inducing luciferase expression 10-fold over the antiestrogen fulvestrant or vehicle. Furthermore, the array captured a dose response to estrogen, demonstrating the sensitivity of bioluminescence quantification. This system provides a tool for basic science research, with potential application for the development of patient specific therapies

Controlled surface-associated delivery of genes and oligonucleotides

A system and methods for controlled gene delivery comprising condensed nucleic acids complexed with polylinkers, wherein the complexes are covalently and/or noncovalently bound to the surface of a substrate capable of supporting cell adhesion. The gene delivery system achieves temporal and spatial control of nucleic acid delivery to a target cell or cells through control of complex density on the surface of the support substrate, and reversibility of the attachment of the polylinker to the support substrate. The system and method of the invention can be used to create spatial patterns of gene expression, and in tissue engineering, high throughput screening, and gene therapy applications. What is claimed is: 1. A method for increasing transgene expression, comprising making a controlled nucleic acid delivery system, said system comprising forming nucleic acid polylinker complexes capable of being delivered to cells cultured on a support substrate, wherein said complexes are formed prior to being covalently or non-covalently immobilized to the surface of a support substrate, and wherein said method comprises: a) contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex, said complex being formed prior to attachment to a support substrate; and b) immobilizing the nucleic acid-polylinker complex to a support substrate; and wherein said cells are added to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate. 2. The method of claim 1, further comprising modification of the support substrate with serum prior to addition of the nucleic acid-polylinker complex, and wherein said modification allows for an increase in transgene expression. 3. The method of claim 1, wherein the extent of transgene expression is dependent upon substrate modification and complex formation. 4. The method of claim 1, wherein said nucleic acid polylinker complexes are polyplexes or lipoplexes. 5. The method of claim 1, wherein said support substrate is polystyrene, gold, hyaluronic acid collagen hydrogels or polylactide-co-glycolide (PLG). 6. The method of claim 2, wherein said substrate modification is made by treatment with serum. 7. The method of claim 1, wherein delivery of the nucleic acid-polylinker complexes to cells occurs from a polystyrene surface treated with serum, and wherein said delivery results in similar or greater percentage of transfected cells relative to bolus delivery. 8. The method of claim 1, said method further comprising release of the nucleic acid from the nucleic acid-polylinker complexes, wherein said release is maximized when the support substrate is treated with serum. 9. A method for increasing transgene expression, comprising making a controlled nucleic acid delivery system, said system comprising forming nucleic acid polylinker complexes capable of being delivered to cells cultured on a support substrate, wherein said complexes are covalently or non-covalently immobilized to the surface of a support substrate, and wherein said method comprises: a) contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex; b) immobilizing the nucleic acid-polylinker complex to a support substrate; and c) adding cells to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate, wherein the release of nucleic acid from the nucleic acid-polylinker complexes is further enhanced when the support substrate containing the complexes is treated with serum or is incubated in conditioned medium. 10. The method of claim 2, wherein the delivery of the nucleic acid-polylinker complexes to cells from a serum-modified support substrate results in higher cellular association of the nucleic acid-polylinker complexes with the support substrate. 11. A method for increasing transgene expression, comprising the steps of: a) making a controlled nucleic acid delivery system by contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex, wherein said complex is formed prior to addition to a support substrate; b) immobilizing the nucleic acid-polylinker complex to a support substrate; wherein said immobilizing is accomplished by covalent or non-covalent means, and c) adding the cells into which transgene expression is desired to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate. 12. The method of claim 11, wherein said support substrate comprises a biodegradable or non-biodegradable material. 13. The method of either one of claim 1 or 11, wherein said complexes are formed prior to attachment to the solid support substrate. 14. The method of claim 12, wherein said biodegradable material is a hydrogel and said non-biodegradable material is polystyrene or gold. 15. The method of claim 12, wherein said hydrogel comprises a mixture of hyaluronic acid and collagen. 16. A method for increasing transgene expression, wherein said method promotes transfection of primary cells, comprising the steps of: a) making a controlled nucleic acid delivery system by contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex; b) immobilizing the nucleic acid-polylinker complex to a support substrate; wherein said immobilizing is accomplished by covalent or non-covalent means, and c) adding the cells into which transgene expression is desired to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate, wherein the biodegradable material is a hydrogel and the non-biodegradable material is polystyrene or gold, and wherein the hvdrogel comprises a mixture of hyaluronic acid and collagen. 17. The method of either one of claim 1 or 11, wherein said nucleic acid polylinker complexes are immobilized to the support substrate using biotin and avidin, or an avidin derivative, or by non-specific adsorption. 18. The method of claim 17, wherein said avidin derivative is streptavidin or neutravidin. 19. The method of any one of claim 11–15, wherein the method further comprises controlling the size of the nucleic acid polylinker complex by regulating the salt content during complex formation. 20. The method of claim 19, wherein controlling the size of said complex formation is accomplished by the presence or absence of salt during the formation of the complexes, wherein the forming of large diameter complexes in the presence of salt results in increased transgene expression, and wherein the forming of small diameter complexes in the absence of salt results in a greater percentage of cells being transfected. 21. The method of claim 19, wherein the salt is sodium chloride. 22. A method for increasing transgene expression, comprising the steps of: a) making a controlled nucleic acid delivery system by contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex; b) immobilizing the nucleic acid-polylinker complex to a support substrate; wherein said immobilizing is accomplished by covalent or non-covalent means, and c) adding the cells into which transgene expression is desired to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate, wherein said method further comprises release of the nucleic acid from the substrate, wherein the release is optimized by using conditioned medium. 23. The method of claim 11, wherein said method further comprises biotinylation of said complex to enhance release of said complex from said substrate. 24. The method of either of claim 1 or 11, wherein the nucleic acid is DNA, RNA or an oligonucleotide. 25. The method of claim 24, wherein said oligonucleotide is an antisense oligonucleotide or a catalytic RNA capable of interfering with the expression of a gene. 26. The controlled nucleic acid delivery system of either of claim 1 or 11, wherein the polylinker is a cationic polymer, cationic lipid, cationic protein, or cationic peptide

Drosophila S2 Cells Are Non-Permissive for Vaccinia Virus DNA Replication Following Entry via Low pH-Dependent Endocytosis and Early Transcription

Vaccinia virus (VACV), a member of the chordopox subfamily of the Poxviridae, abortively infects insect cells. We have investigated VACV infection of Drosophila S2 cells, which are useful for protein expression and genome-wide RNAi screening. Biochemical and electron microscopic analyses indicated that VACV entry into Drosophila S2 cells depended on the VACV multiprotein entry-fusion complex but appeared to occur exclusively by a low pH-dependent endocytic mechanism, in contrast to both neutral and low pH entry pathways used in mammalian cells. Deep RNA sequencing revealed that the entire VACV early transcriptome, comprising 118 open reading frames, was robustly expressed but neither intermediate nor late mRNAs were made. Nor was viral late protein synthesis or inhibition of host protein synthesis detected by pulse-labeling with radioactive amino acids. Some reduction in viral early proteins was noted by Western blotting. Nevertheless, synthesis of the multitude of early proteins needed for intermediate gene expression was demonstrated by transfection of a plasmid containing a reporter gene regulated by an intermediate promoter. In addition, expression of a reporter gene with a late promoter was achieved by cotransfection of intermediate genes encoding the late transcription factors. The requirement for transfection of DNA templates for intermediate and late gene expression indicated a defect in viral genome replication in VACV-infected S2 cells, which was confirmed by direct analysis. Furthermore, VACV-infected S2 cells did not support the replication of a transfected plasmid, which occurs in mammalian cells and is dependent on all known viral replication proteins, indicating a primary restriction of DNA synthesis

Substrate-Mediated Gene Delivery for Assessment of Signal Transduction Pathways in Cancer Cells

Gene delivery has the potential to be used in diagnostic applications, specifically to investigate cellular signal transduction pathways responsible for disease. Analysis of multiple pathways or genes in a parallel format can be achieved using a transfected cell array, a high throughput approach to correlate gene expression with functional cell responses, based on gene delivery from a substrate that supports cell adhesion. Substrate-mediated gene delivery functions by self-assembling DNA with nonviral vectors, resulting in positively charged complexes that can interact with a biomaterial or substrate. Cells cultured on the substrate are exposed to elevated DNA concentrations within the local microenvironment, which enhances transfection. DNA complexes can be immobilized on the substrate through specific interactions introduced through complementary functional groups on the vector and surface or through nonspecific interactions. As surface properties are critical to the efficiency of the surface delivery approach, self-assembled monolayers (SAMs) of alkanethiols on gold were used to study the mechanisms of transfection by complexes nonspecifically immobilized on chemically specific substrates. Surface hydrophilicity and ionization were found to mediate both immobilization and transfection. Additionally, SAMs were used in conjugation with soft lithographic techniques to imprint substrates with specific patterns of SAMs, resulting in patterned DNA complex deposition and transfection

Transfected Cell Arrays for Assessment of Estrogen Receptor Activation in Breast Cancer Cells

Transfected cell arrays represent a high-throughput approach to correlate gene expression with functional cell responses, based on gene delivery from a substrate that supports cell adhesion. These arrays provide the ability to express, in parallel, thousands of exogenous genes in live cells, giving real-time information on cellular physiology and gene function. While there have been advances in transfected cell arrays, improvements are needed to this technology, including increasing the cells types that can be efficiently transfected and developing better quantification and normalization methods. We have created an array using soft lithography techniques to pattern DNA-lipid complex deposition. Specifically, a mold was fabricated by curing polydimethylsiloxane (PDMS) into thin, flat disks. Rods of precise diameters were then used to punch holes into the mold, with diameters ranging from 1 mm to 3 mm. The PDMS mold was oxidized and then reversibly sealed to polystyrene slides. The holes in the mold, termed microwells, served as reservoirs for complex deposition onto the polystyrene slides. After deposition, the PDMS mold was peeled away from the polystyrene slide, which was then rinsed thoroughly. DNA complexes were immobilized on the slide in distinct regions, replicating the pattern of microwells in the PDMS mold. Transfection of cells seeded onto these slides was also confined to the patterns

Controlled surface-associated delivery of genes and oligonucleotides

A system and methods for controlled gene delivery comprising condensed nucleic acids complexed with polylinkers, wherein the complexes are covalently and/or noncovalently bound to the surface of a substrate capable of supporting cell adhesion. The gene delivery system achieves temporal and spatial control of nucleic acid delivery to a target cell or cells through control of complex density on the surface of the support substrate, and reversibility of the attachment of the polylinker to the support substrate. The system and method of the invention can be used to create spatial patterns of gene expression, and in tissue engineering, high throughput screening, and gene therapy applications. What is claimed is: 1. A method for increasing transgene expression, comprising making a controlled nucleic acid delivery system, said system comprising forming nucleic acid polylinker complexes capable of being delivered to cells cultured on a support substrate, wherein said complexes are formed prior to being covalently or non-covalently immobilized to the surface of a support substrate, and wherein said method comprises: a) contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex, said complex being formed prior to attachment to a support substrate; and b) immobilizing the nucleic acid-polylinker complex to a support substrate; and wherein said cells are added to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate. 2. The method of claim 1, further comprising modification of the support substrate with serum prior to addition of the nucleic acid-polylinker complex, and wherein said modification allows for an increase in transgene expression. 3. The method of claim 1, wherein the extent of transgene expression is dependent upon substrate modification and complex formation. 4. The method of claim 1, wherein said nucleic acid polylinker complexes are polyplexes or lipoplexes. 5. The method of claim 1, wherein said support substrate is polystyrene, gold, hyaluronic acid collagen hydrogels or polylactide-co-glycolide (PLG). 6. The method of claim 2, wherein said substrate modification is made by treatment with serum. 7. The method of claim 1, wherein delivery of the nucleic acid-polylinker complexes to cells occurs from a polystyrene surface treated with serum, and wherein said delivery results in similar or greater percentage of transfected cells relative to bolus delivery. 8. The method of claim 1, said method further comprising release of the nucleic acid from the nucleic acid-polylinker complexes, wherein said release is maximized when the support substrate is treated with serum. 9. A method for increasing transgene expression, comprising making a controlled nucleic acid delivery system, said system comprising forming nucleic acid polylinker complexes capable of being delivered to cells cultured on a support substrate, wherein said complexes are covalently or non-covalently immobilized to the surface of a support substrate, and wherein said method comprises: a) contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex; b) immobilizing the nucleic acid-polylinker complex to a support substrate; and c) adding cells to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate, wherein the release of nucleic acid from the nucleic acid-polylinker complexes is further enhanced when the support substrate containing the complexes is treated with serum or is incubated in conditioned medium. 10. The method of claim 2, wherein the delivery of the nucleic acid-polylinker complexes to cells from a serum-modified support substrate results in higher cellular association of the nucleic acid-polylinker complexes with the support substrate. 11. A method for increasing transgene expression, comprising the steps of: a) making a controlled nucleic acid delivery system by contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex, wherein said complex is formed prior to addition to a support substrate; b) immobilizing the nucleic acid-polylinker complex to a support substrate; wherein said immobilizing is accomplished by covalent or non-covalent means, and c) adding the cells into which transgene expression is desired to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate. 12. The method of claim 11, wherein said support substrate comprises a biodegradable or non-biodegradable material. 13. The method of either one of claim 1 or 11, wherein said complexes are formed prior to attachment to the solid support substrate. 14. The method of claim 12, wherein said biodegradable material is a hydrogel and said non-biodegradable material is polystyrene or gold. 15. The method of claim 12, wherein said hydrogel comprises a mixture of hyaluronic acid and collagen. 16. A method for increasing transgene expression, wherein said method promotes transfection of primary cells, comprising the steps of: a) making a controlled nucleic acid delivery system by contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex; b) immobilizing the nucleic acid-polylinker complex to a support substrate; wherein said immobilizing is accomplished by covalent or non-covalent means, and c) adding the cells into which transgene expression is desired to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate, wherein the biodegradable material is a hydrogel and the non-biodegradable material is polystyrene or gold, and wherein the hvdrogel comprises a mixture of hyaluronic acid and collagen. 17. The method of either one of claim 1 or 11, wherein said nucleic acid polylinker complexes are immobilized to the support substrate using biotin and avidin, or an avidin derivative, or by non-specific adsorption. 18. The method of claim 17, wherein said avidin derivative is streptavidin or neutravidin. 19. The method of any one of claim 11–15, wherein the method further comprises controlling the size of the nucleic acid polylinker complex by regulating the salt content during complex formation. 20. The method of claim 19, wherein controlling the size of said complex formation is accomplished by the presence or absence of salt during the formation of the complexes, wherein the forming of large diameter complexes in the presence of salt results in increased transgene expression, and wherein the forming of small diameter complexes in the absence of salt results in a greater percentage of cells being transfected. 21. The method of claim 19, wherein the salt is sodium chloride. 22. A method for increasing transgene expression, comprising the steps of: a) making a controlled nucleic acid delivery system by contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex; b) immobilizing the nucleic acid-polylinker complex to a support substrate; wherein said immobilizing is accomplished by covalent or non-covalent means, and c) adding the cells into which transgene expression is desired to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate, wherein said method further comprises release of the nucleic acid from the substrate, wherein the release is optimized by using conditioned medium. 23. The method of claim 11, wherein said method further comprises biotinylation of said complex to enhance release of said complex from said substrate. 24. The method of either of claim 1 or 11, wherein the nucleic acid is DNA, RNA or an oligonucleotide. 25. The method of claim 24, wherein said oligonucleotide is an antisense oligonucleotide or a catalytic RNA capable of interfering with the expression of a gene. 26. The controlled nucleic acid delivery system of either of claim 1 or 11, wherein the polylinker is a cationic polymer, cationic lipid, cationic protein, or cationic peptide

Gene Delivery Through Cell Culture Substrate Adsorbed DNA Complexes

Efficient gene delivery is a fundamental goal of biotechnology and has numerous applications in both basic and applied science. Substrate-mediated delivery and reverse transfection enhance gene transfer by increasing the concentration of DNA in the cellular microenvironment through immobilizing a plasmid to a cell culture substrate prior to cell seeding. In this report, we examine gene delivery of plasmids that were complexed with cationic polymers (polyplexes) or lipids (lipoplexes) and subsequently immobilized to cell culture or biomaterial substrates by adsorption. Polyplexes and lipoplexes were adsorbed to either tissue culture polystyrene or serum-adsorbed tissue culture polystyrene. The quantity of DNA immobilized increased with time of exposure, and the deposition rate and final amount deposited depended upon the properties of the substrate and complex. For polyplexes, serum modification enhanced reporter gene expression up to 1500-fold relative to unmodified substrates and yielded equivalent or greater expression compared to bolus delivery. For lipoplexes, serum modification significantly increased the number of transfected cells relative to unmodified substrates yet provided similar levels of expression. Immobilized complexes transfect primary cells with improved cellular viability relative to bolus delivery. Finally, this substrate-mediated delivery approach was extended to a widely used biomaterial, poly(lactide-co-glycolide). Immobilization of DNA complexes to tissue culture polystyrene substrates can be a useful tool for enhancing gene delivery for in vitro studies. Additionally, adapting this system to biomaterials may facilitate application to fields such as tissue engineering