AteloGene Systemic Use

Product#: 1391
$800.00
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AteloGene Systemic Use

Cat. No. 1391
Storage 2-10°C

Description
Immediate administration to experimental animals by simple mixing the synthetic siRNA with AteloGene™ Efficient in vivo transfection of siRNA. The effect of RNAi of preventing degradation by RNase persists for a long time. AteloGene™ has no toxicity, and its main component is atelocollagen that demonstrates high biological compatibility. It is possible to choose AteloGene™ Local Use for local administration or AteloGene™ Systemic Use for systemic administration by injection to the tail vein.

Since AteloGene™ Local Use for local administration is gelated in the body, siRNA is securely kept at the administration site Since AteloGene™ Systemic Use for systemic administration is not gelated in the body but circulated in the blood after injection into the tail vein, siRNA is efficiently delivered to the whole body.
This kit is intended for 10 times of administration. 1.Prefilled syringe (filled with ''AteloGene™'') Each syringe is for 5times of administration - 600 μl x 2 syringes 2. 10 x siRNA buffer - 3mL x 1 bottle 3. Sterilized water

Outline

This in vivo transfection kit is designed for delivering siRNA/miRNA into animal tissues, mouse in particular, to introduce the siRNA/miRNA into cells by systemic administrations.

Atelocollagen is positively charged and can thus electrostatically form complexes with nucleic acids when they are mixed together at appropriate concentrations and ratios. The complexes protect nucleic acids from being degraded by nucleases in in vivo.

What is Atelocollagen?

Collagen is an extracellular matrix found in the dermis, ligaments, bones, etc., and accounts for approximately 30% of the total protein in the human body. The most abundant type of collagen is type I collagen, which has a molecular weight of approximately 300 kDa and comprises three polypeptides. 

The molecular structure of collagen is a right-handed triple helix region and telopeptide (non-helical) regions at the N-terminal and C-terminal of the molecule. These telopeptide regions are composed of two α1 chains and one α2 chain. The triple helix region is conserved among species and shows low immunogenicity, while the telopeptide regions exhibit high immunogenicity. Removal of the telopeptide regions by protease treatment produces atelocollagen, which retains the same properties as collagen. We have developed atelocollagen-based medical devices.

Unwinding of the triple helix of collagen and atelocollagen by heat degeneration produces gelatine. Gelatine is a random coil single polypeptide and has high immunogenicity.
Peptides obtained by hydrolysis with strong acids, strong alkalis, or by enzymatic treatment are called hydrolysed collagen. Gelatine and hydrolysed collagen have totally different properties from collagen due to their structural differences compared to collagen.

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Applications

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i.v. administration of nucleic acid

i.p. administration of nucleic acids


Features

  • Prolonged in vivo RNAi effect because atelocollagen, the main component of AteloGene® products, form complexes with siRNA/miRNA.
  • Since AteloGene® products are made from atelocollagen, it is nontoxic.
  • AteloGene® Systemic Use does not form a gel and siRNA/miRNA are efficiently delivered throughout the entire body via the blood circulation following tail vein injection.
Kit Contents
 

AteloGene® Systemic Use prefilled syringe (Including losses during the preparation steps)

600 μL × 2 syringes

10×siRNA buffer
(It can be used for miRNA as well)

3 mL × 1 bottle

Sterilized water

3 mL × 1 bottle

2 mL microtube

2 tubes

Disposable Syringe

2 tubes

18G needle

4 needles

26G needle

2 needles

Instruction manual

1 leaflet


Devices and reagents required other than those in the kit

  • siRNA/miRNA (PAGE- or HPLC-purified grade is recommended.)
  • Container for preparation of siRNA/miRNA solution (sterilized, RNase-free)
  • Cooling device (crushed ice, cold block, etc.)
  • Tube rotator (that can tumble and agitate such as TAITEC RT-5, Bibby scientific SB3, etc.)
  • Pipetter and tips (sterilized, RNase-free)
  • High-speed refrigerated centrifuge
  • Anesthetic

Cat. No.

Product

Package

#1393 for Cosmo Bio customers

#1395 for REPROCELL customers

AteloGene® Systemic Use

1 kit (10 administrations) *


*It can be used more than 10 administrations depending on target tissue because one administration is assumed to be 200μl.For research use only.
Expiration date: 5 years from manufacturing date printed on the box.

Experimental example

Systemic siRNA administration to mouse metastatic tumour model with AteloGene® Systemic Use

1391_fig1.png

After systemic administration of a luciferase siRNA (Luc siRNA) & AteloGene® mixture to metastatic tumors expressing luciferase, luciferase expression was markedly reduced when compared to the control.


Preparation of AteloGene® Local Use & Systemic Use

Preparation instructions

The following preparation steps are applicable to both AteloGene® Local Use and AteloGene® Systemic Use kits. In this section, the numbers ① – ⑦ refer to the ”Kit contents” on product page. Implement measures to secure an RNase-free environment, as far as possible, to avoid the degradation of siRNA/miRNA in advance.

1) Preparation of AteloGene®

Attach the ⑥18G needle to the ①AteloGene® prefilled syringe. Eject the whole amount (600 μL) into the ④Microtube. After ejection, cool the microtube containing “AteloGene®” on ice.

An excess amount of “AteloGene®” is provided in the ①Prefilled syringe so that 600 μL of AteloGene® can be injected into the ④Microtube regardless of losses during the preparation steps.

2) Preparation of siRNA/miRNA solution

Prepare 5-10 μM and 20-40 μM siRNA/miRNA solutions for local and systemic administrations, respectively.

a. Using siRNA/miRNA stock solutions
Adjust the concentration of the siRNA/miRNA solution with ②10×siRNA buffer and ③Sterilized water to a buffer concentration of 1×. Cool on ice.

b. Using lyophilized siRNA/miRNA
Prepare 1×siRNA buffer by diluting ②10×siRNA buffer with ③Sterilized water. Add 1×siRNA buffer to prepare siRNA/miRNA solutions of the desired concentrations. Cool on ice.

Although other buffers such as TE may be used for preparing the siRNA/miRNA solution, ②10×siRNA buffer is recommended for obtaining the best results.

3) Preparation of AteloGene® & siRNA/miRNA complex

While cooling on ice, gently pour 600 μL of the siRNA/miRNA solution onto 600 μL of AteloGene® in the ④Microtube. Slowly rotate and mix the solutions at 4℃ for 20 minutes. To avoid forming bubbles, the rotation speed should be approximately 4 r.p.m. (in the case of a rotator with a diameter of 20 cm).

4) Deformation of bubbles and preparation for administration

Centrifuge the mixture at 10,000 r.p.m. for 1 minute at 4℃ to deform bubbles. Attach the ⑥18G needle to the ⑤Disposable syringe and slowly draw the mixture while avoiding forming bubbles. Replace the needle with the ⑦26G injection needle and keep the syringe refrigerated until immediately before administration.
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References

Systemic administration in Cancer research

  1. Treating metastatic prostate cancer with microRNA-145. Iscaife A, Reis ST, Morais DR, Viana NI, da Silva IA, Pimenta R, Bordini A, Dip N, Srougi M, Leite KRM. Apoptosis. 2018 Aug;23(7-8):388-395. PMID: 29858716
  2. miR-340 suppresses tumor growth and enhances chemosensitivity of colorectal cancer by targeting RLIP76. Zhang LL, Xie FJ, Tang CH, Xu WR, Ding XS, Liang J. Eur Rev Med Pharmacol Sci. 2017 Jun;21(12):2875-2886. PMID: 28682430
  3. Efficient delivery of micro RNA to bone-metastatic prostate tumors by using aptamer-conjugated atelocollagen in vitro and in vivo. Hao Z, Fan W, Hao J, Wu X, Zeng GQ, Zhang LJ, Nie SF, Wang XD. Drug Deliv. 2016;23(3):874-81. PMID: 24892627
  4. MiR675-5p Acts on HIF-1α to Sustain Hypoxic Responses: A New Therapeutic Strategy for Glioma. Lo Dico A, Costa V, Martelli C, Diceglie C, Rajata F, Rizzo A, Mancone C, Tripodi M, Ottobrini L, Alessandro R, Conigliaro A. Theranostics. 2016 May 8;6(8):1105-18. PMID: 27279905
  5. Identification of Akt1 as a potent therapeutic target for oral squamous cell carcinoma. Nakashiro K, Tanaka H, Goda H, Iwamoto K, Tokuzen N, Hara S, Onodera J, Fujimoto I, Hino S, Hamakawa H. Int J Oncol. 2015 Oct;47(4):1273-81. PMID: 26315674
  6. Atelocollagen-mediated intravenous siRNA delivery specific to tumor tissues orthotopically xenografted in prostates of nude mice and its anticancer effects. Yuan Y, Makita N, Cao D, Mihara K, Kadomatsu K, Takei Y. Nucleic Acid Ther. 2015 Apr;25(2):85-94. PMID: 25692652
Systemic administration in other research
  1. Total Flavones of Rhododendron simsii Planch Flower Protect against Cerebral Ischemia-Reperfusion Injury via the echanism of Cystathionine-γ-Lyase-Produced H2S. Chen S, Zhang JH, Hu YY, Hu DH, Gao SS, Fan YF, Wang YL, Jiao Y, Chen ZW. Evid Based Complement Alternat Med. 2018 May 31;2018:8903849. PMID: 29955237
  2. HSF1 deficiency accelerates the transition from pressure overload-induced cardiac hypertrophy to heart failure through endothelial miR-195a-3p-mediated impairment of cardiac angiogenesis. Wang S, Wu J, You J, Shi H, Xue X, Huang J, Xu L, Jiang G, Yuan L, Gong X, Luo H, Ge J, Cui Z, Zou Y. J Mol Cell Cardiol. 2018 Apr 5;118:193-207. PMID: 29626503
  3. Protective Effect of Let-7 miRNA Family in Regulating Inflammation in Diabetes-Associated Atherosclerosis. Brennan E, Wang B, McClelland A, Mohan M, Marai M, Beuscart O, Derouiche S, Gray S, Pickering R, Tikellis C, de Gaetano M, Barry M, Belton O, Ali-Shah ST, Guiry P, Jandeleit-Dahm KA, Cooper ME, Godson C, Kantharidis P. Diabetes. 2017 Aug;66(8):2266-2277. PMID: 28487436
  4. Signal transducer and activator of transcription 5 plays a crucial role in hepatic lipid metabolism through regulation of CD36 expression. Hosui A, Tatsumi T, Hikita H, Saito Y, Hiramatsu N, Tsujii M, Hennighausen L, Takehara T. Hepatol Res. 2017 Jul;47(8):813-825. PMID: 27593674
  5. Activator Protein-1 Decoy Oligodeoxynucleotide Transfection Is Beneficial in Reducing Organ Injury and Mortality in Septic Mice. Imaizumi T, Matsuda N, Tomita K, Palikhe S, Ohashi W, Hattori K, Hattori Y. Crit Care Med. 2018 May;46(5):e435-e442. PMID: 29406423
  6. Deficiency in catechol-o-methyltransferase is linked to a disruption of glucose homeostasis in mice. Kanasaki M, Srivastava SP, Yang F, Xu L, Kudoh S, Kitada M, Ueki N, Kim H, Li J, Takeda S, Kanasaki K, Koya D. Sci Rep. 2017 Aug 11;7(1):7927. PMID: 28801594
  7. MicroRNAs 106b and 222 Improve Hyperglycemia in a Mouse Model of Insulin-Deficient Diabetes via Pancreatic β-Cell  Proliferation. Tsukita S, Yamada T, Takahashi K, Munakata Y, Hosaka S, Takahashi H, Gao J, Shirai Y, Kodama S, Asai Y, Sugisawa T, Chiba Y, Kaneko K, Uno K, Sawada S, Imai J, Katagiri H. EBioMedicine. 2017 Feb;15:163-172. PMID: 27974246
  8. MiR-29a Assists in Preventing the Activation of Human Stellate Cells and Promotes Recovery From Liver Fibrosis in Mice. Matsumoto Y, Itami S, Kuroda M, Yoshizato K, Kawada N, Murakami Y. Mol Ther. 2016 Oct;24(10):1848-1859. PMID: 27480597
  9. Role of Sialidase in Long-Term Potentiation at Mossy Fiber-CA3 Synapses and Hippocampus-Dependent Spatial Memory. Minami A, Saito M, Mamada S, Ieno D, Hikita T, Takahashi T, Otsubo T, Ikeda K, Suzuki T. PLoS One. 2016 Oct 26;11(10):e0165257. PMID: 27783694
  10. MicroRNA-124 inhibits the progression of adjuvant-induced arthritis in rats. Nakamachi Y, Ohnuma K, Uto K, Noguchi Y, Saegusa J, Kawano S. Ann Rheum Dis. 2016 Mar;75(3):601-8. PMID: 25596157
  11. Pivotal Role of Carbohydrate Sulfotransferase 15 in Fibrosis and Mucosal Healing in Mouse Colitis. Suzuki K, Arumugam S, Yokoyama J, Kawauchi Y, Honda Y, Sato H, Aoyagi Y, Terai S, Okazaki K, Suzuki Y, Mizumoto S, Sugahara K, Atreya R, Neurath MF, Watanabe K, Hashiguchi T, Yoneyama H, Asakura H. PLoS One. 2016 Jul 13;11(7):e0158967. PMID: 27410685
  12. Involvement of Hydrogen Sulfide in Endothelium-Derived Relaxing Factor-Mediated Responses in Rat Cerebral Arteries. Wang M, Hu Y, Fan Y, Guo Y, Chen F, Chen S, Li Q, Chen Z. J Vasc Res. 2016 Oct 13;53(3-4):172-185. PMID: 27732975
  13. RNA interference targeting carbohydrate sulfotransferase 3 diminishes macrophage accumulation, inhibits MMP-9 expression and promotes lung recovery in murine pulmonary emphysema. Kai Y, Tomoda K, Yoneyama H, Yoshikawa M, Kimura H. Respir Res. 2015 Dec 9;16:146. PMID: 26646821
  14. Eomesodermin-expressing T-helper cells are essential for chronic neuroinflammation. Raveney BJ, Oki S, Hohjoh H, akamura M, Sato W, Murata M, Yamamura T. Nat Commun. 2015 Oct 5;6:8437. PMID: 26436530
  15. MicroRNA-451 down-regulates neutrophil chemotaxis via p38 MAPK. Murata K, Yoshitomi H, Furu M, Ishikawa M, Shibuya H, Ito H, Matsuda S. Arthritis Rheumatol. 2014 Mar;66(3):549-59. PMID: 24574214

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