Transfection Conditions Research Articles

Functional Interactions between Mldp (LSDP5) and Abhd5 in the Control of Intracellular Lipid Accumulation

Cellular lipid metabolism is regulated in part by protein-protein interactions near the surface of intracellular lipid droplets. This work investigated functional interactions between Abhd5, a protein activator of the lipase Atgl, and Mldp, a lipid droplet scaffold protein that is highly expressed in oxidative tissues. Abhd5 was highly targeted to individual lipid droplets containing Mldp in microdissected cardiac muscle fibers. Mldp bound Abhd5 in transfected fibroblasts and directed it to lipid droplets in proportion to Mldp concentration. Analysis of protein-protein interactions in situ demonstrated that the interaction of Abhd5 and Mldp occurs mainly, if not exclusively, on the surface of lipid droplets. Oleic acid treatment rapidly increased the interaction between Abhd5 and Mldp, and this effect was suppressed by pharmacological inhibition of triglyceride synthesis. The functional role of the Abhd5-Mldp interaction was explored using a mutant of mouse Abhd5 (E262K) that has greatly reduced binding to Mldp. Mldp promoted the subcellular colocalization and interaction of Atgl with wild type, but not mutant, Abhd5. This differential interaction was reflected in cellular assays of Atgl activity. In the absence of Mldp, wild type and mutant Abhd5 were equally effective in reducing lipid droplet formation. In contrast, mutant Abhd5 was unable to prevent lipid droplet accumulation in cells expressing Mldp despite considerable targeting of Atgl to lipid droplets containing Mldp. These results indicate that the interaction between Abhd5 and Mldp is dynamic and essential for regulating the activity of Atgl at lipid droplets containing Mldp.

Rexinoid-induced Expression of IGFBP-6 Requires RARβ-dependent Permissive Cooperation of Retinoid Receptors and AP-1

The synthetic rexinoid bexarotene (Targretin, LGD1069) inhibits the formation of both estrogen receptor-negative and estrogen receptor-positive breast cancer in preclinical models and controls the expression of growth-regulatory biomarkers, such as IGFBP-6 (insulin-like growth factor-binding protein 6), RARbeta, or cyclin D1. In this study, we identified a classical retinoic acid-responsive element in the first intron in the IGFBP-6 gene adjacent to a consensus AP-1 binding site, both elements essential for rexinoid-induced expression of IGFBP-6. In chromatin binding experiments, bexarotene increased the occupancy of the identified enhancer element by RXRalpha, RARbeta, cJun, cFos, and p300. In normal mammary epithelial cells and T47D breast cancer cells, small interfering RNA-mediated knockdown of all RXR isoforms or RARbeta, but not RARalpha or RARgamma alone, blocked the induction of IGFBP-6 by bexarotene. Simultaneous knockdown of RARalpha and RARgamma abrogated both the induction of RARbeta and the up-regulation and secretion of IGFBP-6. The suppression of either RARbeta or cJun by small interfering RNA blocked the recruitment of RXRalpha and cJun to the enhancer. These results demonstrate a novel cooperative interaction between retinoid receptors and AP-1 orchestrated by RARbeta and highlight a novel mechanism by which RARbeta can mediate the cancer-preventive effects of rexinoids.

Targeting of protease 2A genome by single and multiple siRNAS as a strategy to impair CVB3 life cycle in permissive HeLa cells

Dilated cardiomyopathy as a consequence of myocarditis induced by Coxsackieviruses B3 (CVB3) is a worldwide cause of morbidity and death for which current therapies have shown only modest effectiveness. In the present study we tested the efficacy of a novel CVB3 small interfering RNA (siRNA), directed against the protease 2A genome region (siRNA 3626), alone or in combination with another recently described protease 2A siRNA (siRNA 3541). siRNAs (400 nM) were delivered by lipofection to HeLa cells, which were then infected by CVB3 and analyzed. Under optimized transfection conditions, siRNA 3626 markedly improved the viability of CVB3-infected HeLa cells. This was due to a drastic reduction in the amounts of CVB3 genome, viral capsid protein VP1 and vital virions. Finally, the combination of siRNA 3626/3541 did not potentiate the anti-CVB3 effect compared with an equimolar concentration of either siRNA. In conclusion, these data show the potency of siRNA 3626 in downmodulating CVB3 infection together with the lack of any significant transfection-related toxicity. In addition to placing the selected siRNA 3626 among the most effective anti-CVB3 agents so far described, these data fully justify future attempts to further optimize the siRNA-mediated approach to CVB3-induced myocarditis.

DOCK5 and DOCK1 Regulate Caco-2 Intestinal Epithelial Cell Spreading and Migration on Collagen IV

We observed previously that combined small interfering RNAs (siRNAs) targeting CrkII and CrkL, known activators of guanine nucleotide exchange factor DOCK1, strongly inhibit Caco-2 intestinal epithelial cell spreading and migration on collagen IV. DOCK1 siRNA reduced its expression >95% in Caco-2 cells but inhibited spreading much less than combined CrkII/CrkL siRNAs, suggesting that CrkII/CrkL interact with additional DOCK proteins. siRNA targeting DOCK5, a closely related DOCK1 family member, inhibited Caco-2 spreading similarly to DOCK1 siRNA, and the combined siRNAs synergistically inhibited spreading. Similar results were observed in human umbilical vein endothelial cells, and reverse transcriptase PCR demonstrated DOCK5 siRNA reduction of DOCK5 expression in both cell types. Combined DOCK1/DOCK5 siRNAs also inhibited Caco-2 migration and lamellipodial extension. Expression of DOCK5 cDNA, with silent mutations in the siRNA target region allowing expression simultaneously with DOCK5 siRNA, required CrkII/CrkL to restore cell spreading and DOCK5 coimmunoprecipitated with CrkII and CrkL. DOCK5 association with CrkII and CrkL was greatly reduced by mutations in their NH2-terminal SH3 domains. Expression of the DOCK5 COOH-terminal region (Met1738-Gln1870), containing potential Src homology 3 domain-binding proline-rich sites but lacking other functional regions, inhibited Caco-2 spreading and coimmunoprecipitated with CrkL. Coimmunoprecipitation of full-length DOCK5 with CrkL was strongly reduced by deletion of DOCK5 COOH-terminal amino acids 1832-1870. Green fluorescent protein-tagged DOCK5 localized to the membrane of Caco-2 cells spreading on collagen IV. In these studies, we describe human DOCK5 cloning and expression, our results indicating that, along with DOCK1, DOCK5 is an important mediator of CrkII/CrkL regulation of Caco-2 spreading and migration on collagen IV.

312 CONSTRUCTION OF SPECIFICALLY EXPRESSED VECTOR IN MAMMARY GLAND FOR lacS AND ITS TRANSFECTION INTO BOVINE FETAL FIBROBLASTS MEDIATED BY LIPOSOME

This study was designed to optimize conditions for transfection of a mammary gland specific transgene into bovine fetal fibroblasts. Transfection of Sulfolobus solfataricus β-glycosidase gene (lacS) was mediated by liposome. Neomycin resistance (Neor) and enhanced green fluorescent protein (EGFP) gene were used as genetic markers to screen transgenic somatic cells. A 0.92-kb fragment of bovine β-lactoglobulin gene sequence was obtained from bovine genome by PCR amplification and was inserted into the T site of pMD19-Simple T plasmid. 1.49 kb of lacS gene coding sequence was cloned from Sulfolobus solfataricus genome by PCR amplification and inserted into the pUC19 plasmid. The coding sequence of Neor was derived by PCR amplification from pIRES2-EGFP plasmid and inserted into the BamHI/NheI site of pIRES2-EGFP plasmid. The resultant vector (pNIE) contained a Neor and an EGFP gene, which were linked by an internal ribosome entry site sequence downstream of the cytomegalovirus (CMV) promoter. Finally, the vector pNIE was assembled into the pUC19 plasmid, thus creating a pBLI vector, which contained the Neor and EGFP gene regulated by CMV promoter for expression in a non-tissue specific mode and the lacS gene regulated by bovine β-lactoglobulin promoter for specific expression in mammary gland. Bovine fetal fibroblasts (bFF) were isolated from the ear skin of female fetuses at the age of 2 to 3 months. The cells proliferated well and grew normally in culture, with typical fibroblast morphology and growth curve. The effects of different concentrations of transfection and pBLI were compared on the efficiency of transfection. The passage 4 bFF cells at 70 to 80% confluency were transfected in a 24-well culture plate. 2 × 105 cells were cultured in DMEM with 0.5, 0.75, 1.0, 1.5, 2.0, and 2.5 μg of pBLI using transfection (1, 2, 3, 4, 5, and 6 μL) for 48 h, respectively. The transfected cells were cultured for 48 h before adding G418 at concentrations of 200, 300, 400, 500, 600, 700, 800, and 900 μg mL–1 for 14 d, respectively. Positive cell colonies were selected and purified through both the expression of Neor and EGFP gene under a fluorescence microscopy. The selected colonies were propagated in DMEM containing 300 μg mL–1 G418. The results showed that bright green fluorescence could be detected at 48 h after transfection. 1.0 μg of pBLI plasmid and 3 μL of transfection yielded the desirable efficiency of transfection. More transgenic bFF colonies were selected by G418 at the concentration of 800 μg mL–1. In conclusion, a specifically expressed vector in mammary gland for lacS gene was successfully constructed, transfection parameters were developed, and efficient screening measures were established for detecting transgenic somatic cells.

Different effects of therapeutic ultrasound parameters and culture conditions on gene transfection efficiency

To investigate the effect of different therapeutic ultrasound (TUS) parameters and culture conditions on the cell viability and transfection efficiency of human cervical cancer cells (HeLa). HeLa cells were cultured using two different protocols (in suspension or in monolayer). Subsequently, cells were exposed to different TUS intensity (0.4 W/cm2, 1.0 W/cm2, 1.6 W/cm2, 2.2 W/cm2), duty cycle (DC)(10%, 20%, 50%), exposure time (1 min or 3 min). Cell viability was analyzed by flow cytometry. Gene transfection of red fluorescent protein (DsRED) was detected. TUS intensity and duty cycle had a great impact on the overall results (P<0.01). Cell injury were found to increase progressively with intensity (1.6 W/cm2, 2.2 W/cm2) and duty cycle (50%) and cell detachment was accompanied by ultrasound exposure in adherent cells. Results of factorial design showed that the fashion of cell culture and the TUS parameters had interaction (P<0.01). The ideal conditions that cell viability above 80% producing maximum efficiency were noted to be at 1.0 W/cm2 irradiated 3 min with a duty cycle of 20% in cell suspension. TUS parameters and transfection conditions have a great impact on the gene transfection and cell viability. Optimal parameters could enhance cell membrane permeability, which facilitate to delivering the macromolecules into cells.

Expression and functional role of the protein Orai-1 in skeletal myoblasts and myotubes

Expression of channel protein Orai-1 has been demonstrated in cultured skeletal myoblasts and myotubes. In order to evaluate its functional role in Ca2+ transport in these cells we used small interfering RNA (siRNA) targeted against mRNA coding for Orai-1. Optimal conditions for the effective transfection of myoblasts and myotubes were found. In experiments using myotubes from an mdx cell line we have shown that inactivation of mRNA coding Orai-1 results in complete suppression of 45Ca2+ entry induced by thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase. The data obtained indicate that Orai-1 is involved in store-activated Ca2+ entry into skeletal myotubes.

Targeting XIAP for Chemosensitization Purposes: Different Effect with Different Cytotoxic Drugs.

Defects in apoptosis have been implicated in the resistance of cancer cells to a wide variety of anticancer drugs. XIAP, an inhibitor of both intrinsic and extrinsic apoptotic pathways by inhibiting caspases-3, -7 and -9, has been proposed as a good molecular target for enhancing the effects of cytotoxic drugs since:it is widely expressed in human cancer cell lines and human cancer tissues;downregulation of XIAP expression enhanced the effects of chemotherapeutic agents in various cancer cell line models;XIAP expression correlates with prognosis in some cancers, including in acute myeloid leukemia.Small molecules able to inhibit XIAP have been developed. Furthermore, antisense oligonucleotide inhibitors of XIAP (AEG35156) are already in clinical trials. However, the potential chemosensitization effect of XIAP has not been thoroughly explored, both concerning different tumor models and drugs. The purpose of the current study was to investigate if downregulation of XIAP enhances the effects of doxorubicin and of cytarabine, drugs used in the treatment of acute leukemias, in an in vitro model of blastic phase Chronic Myeloid Leukemia, the K562 cell line. The approach was to downregulate XIAP in K562 cells by using:transient transfection with siRNAs andstable transfection with shRNAs cloned into an appropriate vector.Two different siRNAs targeting XIAP were tested, using two different transfection conditions for siRNA delivery. Relatively modest downregulation of XIAP was observed by Western Blot, and the more potent siRNA sequence and transfection conditions were chosen for the chemosensitization studies. Our results show that downregulation of XIAP expression:reduced cellular viability but this effect was not significant;sensitized cells to doxorubicin (IC50 decreased from 78nM to 55nM) but not to cytarabine.To confirm these results, stable cell lines were established by transfection of shRNAs cloned into an appropriate vector. Modest downregulation of XIAP was achieved in the stable cell line when compared to the control shRNA cell line. In these cell lines the results confirmed that downregulation of XIAP sensitizes to doxorubicin (IC50 decreased from 68nM to 50nM) but not to cytarabine. Further analysis of the cells following treatment with the cytotoxic drugs allowed to confirm that both drugs induced programmed cell death by apoptosis (with cleavage of pro-caspase 3) and further reduction of XIAP protein levels, which is probably due to the apoptotic process itself. However, the shRNAs did not provide a better model for studying chemosensitization than the siRNAs. This fact, together with the modest levels of XIAP downregulation, probably reflects that the shRNA sequence corresponds to the siRNA sequence previously used. In conclusion, downregulation of XIAP sensitized K562 cells to doxorubicin but not to cytarabine. This may be partially due to the level of XIAP downregulation obtained but may also be due to the different mechanisms of action of these drugs, indicating that this should be taken into account when considering targeting XIAP for achieving sensitization to cytotoxic drugs.

Effect of Liposome Encapsulated Vesicular Stomatitis Virus Matrix Protein in Hematological Malignancy

Objective: Vesicular Stomatitis Virus (VSV) has shown antitumor effects in many studies, however as an active virus,it has biosafety problems when being used in human body. Matrix protein (M protein) of VSV also has antitumor effects without the existence of other portions of VSV.Recently liposome is widely used as a gene delivery vector. So the present study was to investigate the effect of liposome as a gene delivery vector in hematological malignant cell lines and to investigate the effect of liposome encapsulated Vesicular Stomatitis Virus Matrix protein in hematological malignancy in vitro.Methods: First, Lipofectamine 2000 was selected as a kind of gene delivery vector and its efficacy in different cell lines was identified by transfecting the plasmid DNA encoding GFP gene into suspension cell lines, including K562 cells (human chronic myeloid leukemia), Jurkat cells (human T cell lymphoma), Raji cells (human B cell lymphoma) and both attachment and suspension cell line CZ-1 cells (human multiple myeloma) according to the instruction provided by the Invitrogen company when the ratios of DNA to Lipofectamine2000 were 1:2,1:2.5,1:3,1:4 and 1:5, respectively. Selecting those cell lines was based on that the transfection efficiency of liposome was above 20% and the ratios which could help to receive the greatest efficiency. The human ovarian cancer cell line A2780 was the positive control and the ratio of DNA to Lipofectamine varied from 1:2.5 to 1:3.Secondly, in vitro study, the P-M group was the one in which the plasmid DNA encoding M protein of Vesicular Stomatitis Virus (VSV-M) was transfected into the selected cell lines by the best ratios of DNA to Lipofectamine2000.The groups of Lipofectamine2000- treated (LIPO) , Lipofectamine2000 encapsulated empty plasmid pVAX -treated (P) and untreated one (BLK) were set as the control groups. Morphological changes were observed and the cell growth inhibition rates in different groups were measured by MTT (Methylthiazolyldiphenyl-tetrazolium bromide) method at 24h, 48h and 72h after transfection. The cell apoptosis rates of K562 cells were tested by Flow Cytometry at 48h after transfection.ResultsThe Lipofectamine2000 transfection efficiency in K562 cells could reach up to 50%, while in different conditions the efficiency in CZ-1 was only 20% and that in Raji and jurkat cells was below 10%. The efficiency could also reach 50% in A2780 cells when the ratio was1:3.Study of the effect of liposome encapsulated Vesicular Stomatitis Virus Matrix protein in hematological malignancy: 2.1 K562 and CZ-1 cells: The morphology of K562 and CZ-1 cells in P-M groups was nearly not changed at 24h,48h and 72h after transfection. Futhermore, MTT method showed that, compared with the P and LIPO groups, there were no growth inhibition in K562 and CZ-1 cells at different time points(P>0.05). 2.2 A2780 cells: In the P-M groups, the morphology of A2780 cells was changed ,and a lot of suspension cells were observed in the medium after 48h. Compared with the P and LIPO groups, there was obvious growth inhibition in A2780 cells at different time points (P<0.05).In the P groups, the morphology of A2780 cells was also changed little. Compared with the LIPO group, there was some growth inhibition in A2780 cells at different time points (P<0.05). In the LIPO and BLK groups, these cells had no morphological changes and no growth inhibition.Flow Cytometry showed that there was no difference between the P-M group and P,LIPO groups in K562 cell line in terms of cell apoptosis rates after 48 hours of transfection with VSV-M by means of- liposome(P>0.05).ConclusionsLiposome was not a good choice for the gene transfection in hematological cell lines, including Raji,CZ-1 and Jurkat; however it had good efficacy in K562 and A2780 cell lines.M protein of Vesicular Stomatitis Virus had no obvious inhibition effect in several hematological malignancy cell lines in the current transfection conditions in vitro, while it had good inhibition effect in A2780 cell line. Futhermore, M protein could not induce cell apoptosis in K562 cells in this study.

Identification of Sensitizing Targets to the Hypomethylating Agent 5-Azacytidine in Acute Myeloid Leukemia Cells Using RNAi-Based Functional Genomics Screening

• Hypomethylating agents like 5-Azacytidine (5Aza) have become an effective therapy for myelodysplastic syndromes (MDS) and show promise in acute myeloid leukemia (AML). In AML, complimentary mechanisms including epigenetic silencing of growth controlling genes, i.e. tumor suppressors, and activation of kinases contribute to malignant transformation. In order to enhance the therapeutic potential of epigenetic therapies, we developed a high-throughput RNA interference (HT-RNAi) platform for large-scale transient gene silencing in acute myeloid leukemia cells. This assay allows for the first time to individually silence hundreds or thousands of genes in combination with 5Aza to identify molecular targets whose inhibition enhances the anti-leukemic effect of hypomethylating agents. As part of assay development for HT-RNAi, ten AML cell lines were used to determine the median inhibitory concentration (IC50) of 5Aza for each AML cell lines. Furthermore, the ten cell lines were tested with a panel of cationic lipid transfection reagents at varying weight to volume (wt:vol) ratios to determine the optimal siRNA transfection conditions. Results from these studies identified two AML cell lines TF1 and ML4, which were advanced into kinome-epigenetic RNAi screens.Using a lipid-based method, cells were reverse transfected for 48hrs with 2 different siRNA sequences per gene targeting a total of 572 kinases. After 48hrs, 5Aza at the calculated IC25 was added for an additional 72 hrs and cell proliferation was measured using a luminescence-based assay. Data was background corrected and analyzed using the B-score method to report the strength and statistical significance of growth inhibition compared to controls. A B-score of <−2 indicates statistical significance with p<0.05 (>95% confidence); a B-score <−1.5 provides >87% confidence and was used as lowest cutoff given that screens are focused and contain validated siRNA to kinases. Analysis of two independent RNAi kinome screens, one in TF1 and the other in ML4, in combination with 5Aza, identified six and eleven kinases respectively whose silencing by two different siRNA sequences (2× coverage) potentiated the effects of 5Aza at B-score <−1.5. In ML4 cells 2 kinases were highly significant with a B-score for both siRNA <−2. Six kinases were common targets in both cell lines with significant growth inhibition at a B-score for both siRNA of at least <−1.5 making these kinases potential important modifiers of response to 5Aza.In summary, initial kinome RNAi screens in myeloid cells identified specific kinases as potential sensitizing targets to hypomethylating agents. Moreover, functional genomic RNAi screens provide a fast and attractive approach to identify molecular targets in AML for the rational development of combination therapies with hypomethylating agents as well as other drug classes.

A Conserved Cys-loop Receptor Aspartate Residue in the M3-M4 Cytoplasmic Loop Is Required for GABAA Receptor Assembly

Members of the Cys-loop superfamily of ligand-gated ion channels, which mediate fast synaptic transmission in the nervous system, are assembled as heteropentamers from a large repertoire of neuronal subunits. Although several motifs in subunit N-terminal domains are known to be important for subunit assembly, increasing evidence points toward a role for C-terminal domains. Using a combination of flow cytometry, patch clamp recording, endoglycosidase H digestion, brefeldin A treatment, and analytic centrifugation, we identified a highly conserved aspartate residue at the boundary of the M3-M4 loop and the M4 domain that was required for binary and ternary gamma-aminobutyric acid type A receptor surface expression. Mutation of this residue caused mutant and partnering subunits to be retained in the endoplasmic reticulum, reflecting impaired forward trafficking. Interestingly although mutant and partnering wild type subunits could be coimmunoprecipitated, analytic centrifugation studies demonstrated decreased formation of pentameric receptors, suggesting that this residue played an important role in later steps of subunit oligomerization. We thus conclude that C-terminal motifs are also important determinants of Cys-loop receptor assembly.

Quantitative Optimization of Reverse Transfection Conditions for 384-Well siRNA Library Screening

A necessary step in all small interfering RNA (siRNA) library screens is introduction of the siRNA into cells. We describe the use of a commercially available glyceraldehyde 3-phosphate dehydrogenase enzymatic assay that is capable of simultaneously assessing the efficiency of siRNA delivery into cells and the lipid toxicity. This assay has been modified to work in 384-well plates using reverse transfection. The assay is fast, inexpensive, and quantitative. Conditions identified as optimal using this technique have been employed successfully in library screens.

RNA interference inhibits expression of cyclooxygenase-2 and matrix metalloproteinase-2 in rabbit corneal stromal cells

To research the effect of short hairpin RNA (shRNA) targeting cyclooxygenase-2 (COX-2) on the expression of COX-2 and MMP-2 in rabbit corneal stromal cells in vitro. It was a experimental study. Rabbit corneal stromal cells were cultured in vitro. The transfection efficiency of siRNA mediated by HiperFect Transfection Reagent was determined in rabbit corneal stromal cells in order to optimize the transfection condition. shRNAs (shRNA-1,2,3) specific for COX-2 and one negative control nonspecific shRNA were designed, then were transfected by HiperFect Transfection Reagent into both normal rabbit corneal stromal cells and those which were stimulated by IL-1alpha. 6, 12, 24, 48, 72 hours after IL-1alpha added, cells were collected for Real-Time PCR to detect the gene expression of COX-2 and MMP-2, then the results were compared with those of control group. The data of all groups at the same time was analyzed by one-factor analysis of variance, and the comparison of any two groups was carried out by q-test. The siRNA mediated by HiperFect Transfection Reagent can be transfected efficiently into rabbit corneal stromal cells in vitro, and the maximum efficiency was 70%-80%. The expression of COX-2 and MMP-2 mRNA after IL-1alpha stimulated was much higher than that of blank group. shRNA-2 can significantly inhibit the expression of COX-2 and MMP-2 mRNA in corneal stromal cells stimulated with IL-1alpha. The level of COX-2 and MMP-2 was reduced 83.04% and 73.69% respectively, compared with the expression of single IL-1alpha stimulated group, the difference had statistical significance (q = 24.03, P = 0.00; q = 14.76, P =0.00). The difference of COX-2 mRNA among transfection groups of shRNA-1, shRNA-3, negative control shRNA and single IL-1alpha stimulated group had no statistical significance (F = 0.02, P =0.99). The difference of MMP-2 mRNA among those groups also had no statistical significance (F = 0.02, P = 0.98). The RNA interference targeting COX-2 can effectively inhibit the expression of COX-2 and MMP-2 in IL-1alpha stimulated rabbit corneal stromal cells in vitro.

Niemann-Pick C1 Functions in Regulating Lysosomal Amine Content

Mutations in the late endosomal/lysosomal membrane protein Niemann-Pick C1 (NPC1) are known to cause a generalized block in retrograde vesicle-mediated transport, resulting in the hyper-accumulation of multiple lysosomal cargos. An important, yet often overlooked, category of lysosomal cargo includes the vast array of small molecular weight amine-containing molecules that are substrates for ion trapping in the highly acidic organelle lumen. We show here that the introduction of amine-containing molecules in lysosomes can significantly stimulate NPC1-mediated late endosome/lysosome fusion, and subsequently the secretion of lysosomal cargo. To illustrate the physiological importance of this NPC1-mediated transport pathway, we show that NPC1-deficient cells are more susceptible to the toxic effects of a lysosomotropic polyamine metabolite 3-aminopropanal. Moreover, NPC fibroblasts are shown to have higher levels of polyamine oxidase, an enzyme involved in the formation of 3-aminopropanal. Collectively, these findings provide strong support for a novel functional role for NPC1 and may also provide clues toward understanding NPC disease progression.

Optimization of Procedure for Efficient Gene Transfer into Porcine Somatic Cells with Lipofection

The objective of this study was to establish conditions for transfection of a foreign gene into somatic cells using cationic lipid reagents and to evaluate the effects of transfection on in vitro development of somatic cell nuclear transfer (SCNT) embryos. Green fluorescent protein (GFP) gene was used as a foreign gene and a non-transfected somatic cell was utilized as a control karyoplast. Monolayers of porcine cells were established and subsequently transfected with a GFP-expressing gene (pEGFP-Nl) using three types of transfection reagents (LipofectAMINE PLUS, FuGENE 6 or ExGen500). Donor cells used for SCNT included transfected fetal or adult fibroblasts and oviduct epithelial cells, either serum-fed or serum-starved. Oocytes matured in vitro for 42 h were reconstructed with either transfected or non-transfected porcine somatic cells by electric fusion and activation using a single DC pulse of 1.8 kV/cm for 30 μs in Ca 2+ and Mg 2+ -containing 0.26 M mannitol solution. Reconstructed oocytes were subsequently cultured in NCSU-23 medium for 168 h and the developmental competence and cell number in blastocyst were compared. There were no significant differences (P>0.05) in fusion, cleavage rates or development to the blastocyst stage between non-transfected, transfected, serum-fed and serum-starved cells. However, the rates of GFP-expressing blastocysts were higher in the FuGENE 6 group (71.4%) among transfection reagents and in the fetal fibroblasts group (70.4%) for donor cells. These results indicate that fetal fibroblasts transfected with FuGENE 6 can be used as donor cells for porcine SCNT and that GFP gene can be safely used as a marker of foreign genes in porcine transgenesis.

The Role of TRAF2 Binding to the Type I Interferon Receptor in Alternative NFκB Activation and Antiviral Response

Type I interferons (IFNs) play critical roles in the host defense by modulating gene expression through the IFN-dependent activation of STAT and NFkappaB transcription factors. Previous studies established that IFN activates NFkappaB through a classical NFkappaB pathway that results in IkappaBalpha degradation and formation of p50-containing NFkappaB complexes, as well as an alternative pathway that involves NFkappaB-inducing kinase and TRAF2, which results in the formation of p52-containing NFkappaB complexes. In this study, we examined the interaction of TRAF proteins with the type I IFN receptor. We found that TRAF2 was directly coupled to the signal-transducing IFNAR1 subunit of the IFN receptor. By immunoprecipitation, overexpression of epitope-tagged IFNAR1 constructs, and glutathione S-transferase pulldown experiments, we demonstrate that TRAF2 rapidly binds to the IFNAR1 subunit of the IFN receptor upon IFN binding. The membrane proximal half of the IFNAR1 subunit was found to directly bind TRAF2. Moreover, analysis of mouse embryo fibroblasts derived from TRAF2 knock-out mice demonstrated that TRAF2 plays a critical role in the activation of the alternative NFkappaB pathway by IFN, but not the classical NFkappaB pathway, as well as in the antiviral action of IFN. Our results place TRAF2 directly in the signaling pathway transduced through the IFNAR1 subunit of the IFN receptor. These findings provide an important insight into the molecular mechanisms by which IFN generates signals to induce its biological effects.

Phospholipase C-δ1 Expression Is Linked to Proliferation, DNA Synthesis, and Cyclin E Levels

Phospholipase C-δ1 Expression Is Linked to Proliferation, DNA Synthesis, and Cyclin E Levels

Gene expression silencing with ‘specific’ small interfering RNA goes beyond specificity – a study of key parameters to take into account in the onset of small interfering RNA off‐target effects

RNA-mediated gene silencing (RNA interference) is a powerful way to knock down gene expression and has revolutionized the fields of cellular and molecular biology. Indeed, the transfection of cultured cells with small interfering RNAs (siRNAs) is currently considered to be the best and easiest approach to loss-of-function experiments. However, several recent studies underscore the off-target and potential cytotoxic effects of siRNAs, which can lead to the silencing of unintended mRNAs. In this study, we used a low-density microarray to assess gene expression modifications in response to five different siRNAs in various cell types and transfection conditions. We found major differences in off-target signature according to: (a) siRNA sequence; (b) cell type; (c) duration of transfection; and (d) post-transfection time before analysis. These results contribute to a better understanding of important parameters that could impact on siRNA side effects in knockdown experiments.

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.

Synthetic nanoparticle vehicles deliver siRNA to vascular endothelial cells for VCAM‐1 knockdown

The therapeutic promise of siRNA relies on its ability to knock down genes with minimal side effects. Upregulation of inflammatory genes such as vascular cell adhesion molecule‐1 (VCAM‐1) in atherosclerosis suggests that targeted delivery of siRNA as an antiatherosclerotic therapy might modify disease progression. We have shown previously that liquid perfluorocarbon nanoparticle emulsions (PFC‐NP) can serve as molecularly targeted vehicles for imaging and drug delivery in vivo. In this study, we sought to utilize PFC NP as carriers of siRNA to endothelial cells. Cationic PFC‐NP emulsions were produced with 1,2‐Dioleoyl‐3‐Trimethylammonium‐Propane (DOTAP). siRNA to VCAM‐1 was complexed to the emulsions and incubated with mouse 2F‐2B endothelial cells. We observed that fluorescently labeled siRNA in cells 24 h after transfection as determined by confocal microscopy. Optimized transfection conditions (10 pM PFC‐NP with 5 nM siRNA) produce a 52% decrease in VCAM‐1 mRNA levels after 48h. Corresponding viability data measured 24 h after transfection using flow cytometry indicated 80–90% viability in 2F‐2B cells under similar transfection conditions. Accordingly, these particles offer an efficient method for siRNA delivery to endothelial cells and they can simultaneously serve as a vehicle for molecular imaging to confirm siRNA delivery. This work was supported by NIH grant #HL073646