Browsing by Subject "siRNA"

Now showing 1 - 4 of 4
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    Chitosan/interfering RNA nanoparticle mediated gene silencing in disease vector mosquito larvae
    (JOVE, 2015-03-25) Zhang, Xin; Mysore, Keshava; Flannery, Ellen; Michel, Kristin; Severson, David W.; Zhu, Kun Yan; Duman-Scheel, Molly; Department of Medical and Molecular Genetics, IU School of Medicine
    Vector mosquitoes inflict more human suffering than any other organism-and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.
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    Exploring Chondrocyte Integrin Regulation of Growth Factor IGF-I Expression from a Transient pAAV Vector
    (2013-08-20) Ratley, Samantha Kay; Trippel, Stephen B.; Lin, Chien-Chi; Stocum, David L.
    Insulin-like Growth Factor I (IGF-I) is a growth factor that stimulates both mitogenic and anabolic responses in articular chondrocytes. While it has been shown that exogenous IGF-I can regulate chondrocyte integrins, little is known regarding regulatory effects of IGF-I produced from a transiently expressed plasmid based adeno-associated virus (pAAV) vector. Because chondrocytes are using cellular machinery to overexpress IGF-I, it is of interest to see whether or not pAAV IGF-I will significantly upregulate or downregulate chondrocyte integrins. Additionally, it is of interest to know whether chondrocyte adhesion through integrins will have any regulatory effects on the production of IGF-I from the transgene. Therefore, this study will ascertain if pAAV IGF-I will have similar effects that exogenous IGF-I has on integrin regulation and if integrin silencing mechanisms will affect the production of IGF-I from the transgene. To test these hypotheses, adult articular chondrocytes were doubly transfected with the pAAV vector for IGF-I and short interference ribonucleic acid (siRNA) for integrins beta 1 and alpha V. Gene products were monitored at the transcriptional levels using quantitative real time polymerase chain reactions (qPCR) and IGF-I protein production was monitored at the translational level using enzyme linked immunoabsorbant assays (ELISAs). Adult articular chondrocytes doubly transfected were encapsulated in a three dimensional hydrogel system to simulate an in vivo environment. Samples were collected for analysis at days 2, 4, and 6 post encapsulation. Results show that IGF-I treatment with the pAAV vector does not cause significant changes in the transcriptional regulation of the beta 1 integrin in a three dimensional hydrogel system. The pAAV IGF-I vector did not cause significant regulatory changes on integrin alpha V at any time point during the experiment. Additionally, by knocking down the expression levels of integrins by using siRNA, it was shown that integrin knockdown does not have a significant regulatory effect on transcriptional or translational expression levels of IGF-I from the pAAV vector.
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    In Vivo siRNA Delivery and Rebound of Renal LRP2 in Mice
    (Hindawi Publishing Corporation, 2017) Eadon, Michael T.; Cheng, Ying-Hua; Hato, Takashi; Benson, Eric A.; Ipe, Joseph; Collins, Kimberly S.; De Luca, Thomas; El-Achkar, Tarek M.; Bacallao, Robert L.; Skaar, Todd C.; Dagher, Pierre C.; Medicine, School of Medicine
    siRNA stabilized for in vivo applications is filtered and reabsorbed in the renal proximal tubule (PT), reducing mRNA expression transiently. Prior siRNA efforts have successfully prevented upregulation of mRNA in response to injury. We proposed reducing constitutive gene and protein expression of LRP2 (megalin) in order to understand its molecular regulation in mice. Using siRNA targeting mouse LRP2 (siLRP2), reduction of LRP2 mRNA expression was compared to scrambled siRNA (siSCR) in mouse PT cells. Mice received siLRP2 administration optimized for dose, administration site, carrier solution, administration frequency, and administration duration. Kidney cortex was collected upon sacrifice. Renal gene and protein expression were compared by qRT-PCR, immunoblot, and immunohistochemistry (IHC). Compared to siSCR, siLRP2 reduced mRNA expression in PT cells to 16.6% ± 0.6%. In mouse kidney cortex, siLRP2 reduced mRNA expression to 74.8 ± 6.3% 3 h and 70.1 ± 6.3% 6 h after administration. mRNA expression rebounded at 12 h (160.6 ± 11.2%). No megalin renal protein expression reduction was observed by immunoblot or IHC, even after serial twice daily dosing for 3.5 days. Megalin is a constitutively expressed protein. Although LRP2 renal mRNA expression reduction was achieved, siRNA remains a costly and inefficient intervention to reduce in vivo megalin protein expression.
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    siRNA Targeted to p53 Attenuates Ischemic and Cisplatin-Induced Acute Kidney Injury
    (2009-08) Molitoris, Bruce A.; Dagher, Pierre C.; Sandoval, Ruben M.; Campos, Silvia B.; Ashush, Hagit; Fridman, Eduard; Brafman, Anat; Faerman, Alexander; Atkinson, Simon J.; Thompson, James D.; Kalinski, Hagar; Skaliter, Rami; Erlich, Shai; Feinstein, Elena
    Proximal tubule cells (PTCs), which are the primary site of kidney injury associated with ischemia or nephrotoxicity, are the site of oligonucleotide reabsorption within the kidney. We exploited this property to test the efficacy of siRNA targeted to p53, a pivotal protein in the apoptotic pathway, to prevent kidney injury. Naked synthetic siRNA to p53 injected intravenously 4 h after ischemic injury maximally protected both PTCs and kidney function. PTCs were the primary site for siRNA uptake within the kidney and body. Following glomerular filtration, endocytic uptake of Cy3-siRNA by PTCs was rapid and extensive, and significantly reduced ischemia-induced p53 upregulation. The duration of the siRNA effect in PTCs was 24 to 48 h, determined by levels of p53 mRNA and protein expression. Both Cy3 fluorescence and in situ hybridization of siRNA corroborated a short t½ for siRNA. The extent of renoprotection, decrease in cellular p53 and attenuation of p53-mediated apoptosis by siRNA were dose- and time-dependent. Analysis of renal histology and apoptosis revealed improved injury scores in both cortical and corticomedullary regions. siRNA to p53 was also effective in a model of cisplatin-induced kidney injury. Taken together, these data indicate that rapid delivery of siRNA to proximal tubule cells follows intravenous administration. Targeting siRNA to p53 leads to a dose-dependent attenuation of apoptotic signaling, suggesting potential therapeutic benefit for ischemic and nephrotoxic kidney injury.