Thursday, May 9, 2013

i-Fect Delivers IGF1 siRNA to the Mouse Brain

from Pete Shuster at Neuromics
 
Intraventricular Injections Used to Study Neuronal Survival During Post Natal Development

Neuromics’ i-FectTM Transfection Kit continue to be successfully used to deliver siRNA, shRNA and miRNA to cell cultures and the CNS in vivo (via intrathecal, epidural and intraventricular injection). Genes studied include: ABCA, ASIC, β-arrestin, CAV1.2, IGF1, CX3CR1, DOR, ELOVL4, IKBKAP, K+-ATPase, KV1.1, KV9.1 ,The β3 subunit of the Na+,K+-ATPase, NTS1, NAV1.8, NTS1, NOV, Raf-1, RANK,SNSR1, hTertTRPV1 NOV, Survivin, TLR4, Troy and TRPV1. Related Publications.

It is always an honor for one our products to be referenced in one of the Nature publications. Here researchers use i-Fect to study the impact of microglial derived IGF1 silencing on Neuronal Survival: Masaki Ueno,Yuki Fujita, Tatsuhide Tanaka, Yuka Nakamura, Junichi Kikuta, Masaru Ishii, Toshihide Yamashita. Layer V cortical neurons require microglial support for survival during postnatal development. Nature Neuroscience 16, 543–551 (2013) doi:10.1038/nn.3358. …vehicle (PBS) was delivered intraventricularly through the cisterna magna with a glass pipette while P3 mice were cold anesthetized. Igf1 siRNA (stealth siRNA, Invitrogen) or Igfbp5 siRNA with i-Fect reagent (Neuromics) was delivered intraventricularly through the cisterna magna at P3 twice with a 12-h interval

Images: (a) Igf1 expression (blue) and Iba1-positive microglia (brown) in P5 brain. Scale bar represents 400 μm. (b) Magnified view of dotted square in a. Scale bar represents 50 μm. (c,d) IGF1Ra expression (red) in CTIP2-positive (c) and SATB2-positive (d) layer V neurons at P5. Scale bar represents 50 μm. (e) IGF1 protein levels in medium from cultured cortical neurons, microglia and neurons with microglia in transwell systems detected by enzyme-linked immunosorbent assay (ELISA). **P < 0.01 (neuron, microglia + neuron, n = 5; microglia, n = 6 experiments; one-way ANOVA followed by Tukey-Kramer test). (f) The number of cleaved caspase-3–positive neurons in transwell systems treated with LY294002 or H-1356 or transfected to microglia with Igf1 siRNA. *P < 0.05, **P less than  0.01 (n = 3 experiments, one-way ANOVA followed by Tukey-Kramer test). (g) Neuronal phospho-AKT expression in cultured cortical neurons and those with microglia in transwell system. (h) TUNEL-positive cells in H-1356–treated cortex (36 h after treatment). Scale bar represents 100 μm. (i) The number of TUNEL-positive apoptotic cells in each layer in vehicle- (phosphate-buffered saline, PBS) or H-1356–treated mice (36 h after injection). *P < 0.05 (n = 4 brains, one-way ANOVA followed by Tukey-Kramer test). (j) Cleaved caspase-3–labeled cells (red) expressing CTIP2 (green, arrowheads). Scale bar represents 100 μm. (k,l) TUNEL-positive cells in the cortex treated with control or Igf1 siRNA (48 h after treatment). Scale bar represents 100 μm. (m) The number of TUNEL-positive apoptotic cells in each layer in control siRNA– and Igf1 siRNA–treated mice. **P less than 0.01 (n = 5 brains, one-way ANOVA followed by Tukey-Kramer test). Error bars represent s.e.m.


 

Related Products

i-Fect

siRNA Transfection Kit  MANUAL
Catalog Number Size

NI35150 1 x 0.75 ml

NI35750 5 x 0.75 ml


Type: Cationic Lipid
Description/Data:
Picture


Figure:  siRNA-mediated suppression of target gene expression in Schwann cells. A, Detergent extracts of siNeg- or siGly1-transfected Schwann cells were digested with heparitinase and subjected to immunoblot analysis with anti-glypican-1 antibodies (top); aliquots of undigested extracts were immunoblotted with anti-actin antibodies (bottom) to verify equal sample loading. B, Cell surface expression of glypican-1 was assessed by immunofluorescent staining of transfected cells 48 h after transfection using anti-glypican-1 antibodies (green) and DAPI (4′,6′-diamidino-2-phenylindole) to stain nuclei (red). C, Schwann cells were transfected with siNeg or si alpha4(V), and conditioned media and cell lysates were harvested 48 h later (left) or at the indicated times after transfection (right); aliquots of medium (top) or cell lysates (bottom) were subjected to immunoblot analysis and stained with anti-alpha4(V) collagen (top) or anti-actin (bottom) antibodies.

i-FectTM is a novel cationic lipid formulation specifically designed for efficient delivery of siRNAs (small interfering RNAs) into a wide variety of cell types.  Customers have been using i-Fect for both in vitro and in vivo applications.

Kit Contents
  • Hydrated i-Fect ™ Lipid
  • siRNA Diluent
     

Protocol


 

i-Fect ™ Hi Put 96-New

i-Fect based system for high throughput delivery of siRNAs (small interfering RNAs) into a wide variety of cell types.
Picture

Image: 96 well plate showing initial dilution series for optimizing siRNA delivery.
i-Fect ™ has proven to be an effective tool for delivering siRNA and DsiRNA in vitro and in vivo. These capablities are confirmed in a growing list of Publications referencing i-Fect.
i-Fect  Hi-Put 96 provides a convenient high-throughput format for optimizing transfection conditions and performing siRNA transfection in 96-well plates. All wells (except those in Column 12) contain a predetermined amount of the i-Fect  siRNA Transfection Reagent, a cationic lipid based reagent that has been extensively screened in many mammalian cell lines in order to achieve:
  • Optimal dilutions for high percentage delivery of siRNA
  • Functional gene silencing post siRNA delivery.
  • Compatibility with diverse growth conditions (with and without serum).
  • Low cytotoxicity.

Name CatalogUE Type Species Applications Size
96 Well Dilution Series PL30001 Cationic Lipid

96 Well Tritration Plate
4X96 Well Tritration Plate

96 Well (High) PL30002 Cationic Lipid

96 Well Plate-High
4X96 Well Plate-High

96 Well (Low) PL30003 Cationic Lipid

96 Well Plate (Low)
4X96 Well Plate (Low)

siRNA-intact SI35100 RNAse Inhibitor

100 units in 100 ul


Where can I find more information about Neuromics?

Visit the manufacturer page at www.stratech.co.uk/neuromics, email info@stratech.co.uk or call +44 (0) 1638 782600.

Stratech Scientific is a distributor of high quality, competitively priced, reliable products for research laboratories throughout the UK and Europe.

Please contact us to find out which product ranges we can supply in your country.
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