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NANOPARTICLE-based In Vivo Transfection Kit
Small RNA (siRNA, microRNA, mRNA) and plasmid DNA in vivo delivery reagent
Modes of administration:
- Systemic intravenous (i.v.) injection
- Direct intratumoral (i.t.) injection
Altogen’s NANOPARTICLE In Vivo Transfection Reagent
- Efficient delivery to the brain, heart, lung, liver, pancreas, kidney, and multiple tumor types via tail vein administration
- Functionally tested in mice (BALB/c, Nude, NOD/SCID) and Sprague Dawley rats
- Complexes are stable in serum for 16 hours. Applicable for plasmid DNA and siRNA co-injection
- Efficient siRNA and pDNA delivery via direct subcutaneous tumor injection (various tumor types)
- Minimal toxicity. No significant change in cytokines expression and other safety biomarkers were observed
- Download NANOPARTICLE-based in vivo transfection protocol: [PDF] [Word]
- Download PowerPoint presentation for NANOPARTICLE-based in vivo transfection kit: [PPT]
- Download safety data sheet: [PDF]
- UPC/GTIN/EAN: 860002089702
- Brand: ALTOGEN®, developed and manufactured by Altogen Biosystems
DATA
Brain transfection. Delivery of RNA and DNA biomolecules into the mouse brain tissue and glioblastoma brain tumor.
Figure 1. Systemic administration (i.v.) of Nanoparticle In Vivo Transfection Reagent conjugated with 80 ug of chemically modified siRNA targeting Lamin A/C mRNA or scrambled sequence non-silencing siRNA control (or pDNA expression vector encoding Lamin A) and following the recommended transfection protocol. Nanoparticle conjugated RNA/DNA complexes were injected at constant pressure into the tail veins of NOD/SCID mice (orthotopic glioblastoma xenograft model developed by Altogen Labs). Following 72 hours post first injection, the brain and brain tumor tissues were homogenized and lysed in RIPA Buffer supplemented with protease inhibitor cocktail. High sensitivity BCA protein assay was used to normalize the protein concentration from each individual sample. Quantitative immunoblotting was performed to analyze for the change in Lamin A expression levels using the automated western blot system WES (Protein Simple; San Jose, CA). Images acquired with the contrast set to white −100 and black 4000 for standardization. Mice treated with scrambled non-silencing siRNA served as controls. Statistical data analysis were conducted using Compass software. Technical replicates (n=10). Biological replicates (n=5). P-value < 0.01
Figure 2. Intravenous administration of Nanoparticle In Vivo Transfection Reagent conjugated with 80 ug of chemically modified siRNA targeting Lamin A/C mRNA or scrambled sequence non-silencing siRNA control (or pDNA expression vector encoding Lamin A) and following the recommended transfection protocol. Nanoparticle conjugated RNA/DNA complexes were injected at constant pressure into the tail veins of NOD/SCID mice. Following 72 hours post first injection, the spleen and kidney tissues were homogenized and lysed in RIPA Buffer supplemented with protease inhibitor cocktail. High sensitivity BCA protein assay was used to normalize the protein concentration from each individual sample. Quantitative immunoblotting was performed to analyze for the change in expression of Lamin A using the automated western blot WES system. Images acquired with the contrast set to white −100 and black 4000 for standardization. Statistical data analysis were conducted using Compass software. Technical replicates (n=10). Biological replicates (n=5). P-value < 0.01
Figure 3. Intravenous administration of Nanoparticle In Vivo Transfection Reagent conjugated with 80 ug of chemically modified siRNA targeting Lamin A/C mRNA or scrambled sequence non-silencing siRNA control (or pDNA expression vector encoding Lamin A) and following the recommended transfection protocol. Nanoparticle conjugated RNA/DNA complexes were injected at constant pressure into the tail veins of NOD/SCID mice. Following 72 hours post first injection, the lung and heart tissues were homogenized and lysed in RIPA Buffer supplemented with protease inhibitor cocktail. High sensitivity BCA protein assay was used to normalize the protein concentration from each individual sample. Quantitative immunoblotting was performed to analyze for the change in expression of Lamin A using the automated western blot WES system. Images acquired with the contrast set to white −100 and black 4000 for standardization. Statistical data analysis were conducted using Compass software. Technical replicates (n=10). Biological replicates (n=5). P-value < 0.01
Figure 4. Intravenous administration of Nanoparticle In Vivo Transfection Reagent conjugated with 80 ug of chemically modified siRNA targeting Lamin A/C mRNA or scrambled sequence non-silencing siRNA control (or pDNA expression vector encoding Lamin A) and following the recommended transfection protocol. Nanoparticle conjugated RNA/DNA complexes were injected at constant pressure into the tail veins of NOD/SCID mice. Following 72 hours post first injection, the liver and pancreas tissues were homogenized and lysed in RIPA Buffer supplemented with protease inhibitor cocktail. High sensitivity BCA protein assay was used to normalize the protein concentration from each individual sample. Quantitative immunoblotting was performed to analyze for the change in expression of Lamin A using the automated western blot WES system. Images acquired with the contrast set to white −100 and black 4000 for standardization. Statistical data analysis were conducted using Compass software. Technical replicates (n=10). Biological replicates (n=5). P-value < 0.01
Figure 5. Systemic administration (i.v.) of Nanoparticle-based In Vivo reagent conjugated with siRNA targeting Lamin A/C mRNA or non-silencing control siRNA following the recommended protocol. Tissues were collected and RNA isolated 48 hours after post first injection. Samples were analyzed by qRT-PCR for Lamin A/C gene expression levels. Ribosomal RNA levels were used to normalize the Lamin A/C data. Data are means ± SD (n=6).
ALTOGEN® IN VIVO Transfection Kits supplied with ready-to-run transfection protocols that eliminate the need for extensive transfection optimization experiments. Read more about transfection technology at Altogen’s Transfection Resource.
Nanoparticle Transfection Reagent citation references:
- Nature Biotechnology. 2011 29(4):341-5. Delivery of siRNA to the mouse brain by … Alvarez-Erviti et al [PDF]
- Cardiovascular Research. 2016. 110(1):30-39. Modulators of right ventricular apoptosis and contractility in a rat model of pulmonary hypertension. Zungu-Edmondson et al et al [PDF]
- Hypertension 2015. 65(6):1307-15. Hypoxia-independent upregulation of placental hypoxia inducible factor-1 gene expression … Iriyama T et al [PDF]
- Diabetologia. 2015 Aug; 58(8): 1949–1958. Silencing of miR-195 reduces diabetic cardiomyopathy in C57BL/6 mice. Zheng et al [PDF]
- J Mol Cell Cardiol. 2015 Jan; 0: 174–185. Netrin-1 Abrogates Ischemia Reperfusion-induced Cardiac Mitochondrial Dysfunction via Nitric Oxide-dependent Attenuation of NOX4 Activation and Recoupling of NOS. Siu et al [PDF]
- J Cereb Blood Flow Metab. 2017 Jul;37(7):2359-2367. Inhibition of Src family kinases improves cognitive function after intraventricular hemorrhage or intraventricular thrombin. Liu et al [PDF]
- Nature Medicine. 2016 22, 1131–1139. The long noncoding RNA Chaer defines an epigenetic checkpoint in cardiac hypertrophy. Wang et al [PDF]
Nanoparticle In Vivo Transfection Reagent
Altogen Labs Preclinical Research Services:
Altogen Labs provides GLP compliant CRO services for preclinical research, IND applications, and drug development. Biology contract research services includes over 90 in-house validated xenograft models), development of stable cell lines in just 28 days, ELISA assay development, cell-based and tissue targeted RNAi studies, safety pharm/tox assays, and many other research laboratory studies (both efficacy and safety).
Volume Options:
- 0.5 ml – 10 injections (Catalog #5030)
- 1.5 ml – 30 injections (Catalog #5031)
- 8.0 ml – 160 injections (Catalog #5032)
- 25 ml – 50 rat injections or 500 mouse injections (Catalog #5033)
AltogenBiosystems是一家开发和制造用于生命科学研究,药物发现和开发的转染试剂盒的生物技术公司。转染试剂盒针对特定癌细胞系和原代细胞培养进行了优化,可将生物分子有效递送到靶组织中。通过先进的试剂配方和优化的转染方案实现体外(癌细胞系)和体内(动物组织靶向试剂、癌细胞系)递送货物分子,包括质粒DNA,各种类型的RNA(mRNA,siRNA,shRNA,microRNA),蛋白质和小分子研究。
Altogen生命科学公司致力于研发,生产和销售特定细胞系的转染试剂,用于细胞间生物分子的传递,并通过对转染试剂类型的设计将siRNA和质粒DNA有效地转入不同的细胞系和原代细胞内。Altogen公司开发的聚合物,脂质体,纳米粒子为基础的转染技术分别针对分子生物学,组合化学,和细胞生物学而分别应用。Altogen定制服务提供符合GLP要求定制研究服务,包括代稳定的细胞系,细胞银行和冷冻保存,焦磷酸测序,克隆,RNA干扰(RNAi)和基因沉默服务,发展分析,siRNA文库筛选,并转染服务。稳定的肿瘤细胞株和原代细胞的产生,可以是非常昂贵和费时。该公司的细胞培养科学家的细胞株的选择,无论是利息或shRNA表达载体的稳定表达的基因改造。标准的RNAi技术服务,包括设计与合成的siRNA的利益,验证siRNA的沉默效率,siRNA转染条件的优化,使高效的基因沉默细胞系或原代培养细胞的靶基因。转染培养细胞的瞬时或稳定的引入外源性分子和遗传物质(即RNA或DNA),通常是在生物实验室用来研究基因功能,基因表达的调节,生化映射,突变分析,和蛋白质的生产。科学家利用各种载体分子,这种分子,使质粒DNA(PDNA),信使RNA(mRNA),短干扰RNA(siRNA),小分子RNA(miRNA)的,并进入肿瘤细胞株和原代细胞的蛋白质的基因交付。不幸的是,无单提货的方法或转染试剂,可以适用于所有类型的细胞,细胞的细胞毒性和转染效率显着不同,取决于试剂,协议,并正在利用细胞类型。Altogen生物系统公司提供超过60种类型的细胞的预优化转染试剂盒。纳米粒子,脂质和聚合物基ALTOGEN®在体内转染试剂,使交付功能的RNA和DNA分子在体内。PEG脂质体在体内输送系统减少由于PEG修饰的先天免疫反应,并提供高效的siRNA转染的DNA,并在体内的蛋白质。由科学“杂志(2010年12月17日):PEG脂质体在体内转染试剂盒siRNA的特色Altogen生物系统功能的特定细胞系转染试剂盒
120+细胞转染试剂和活体组织靶向试剂盒制造商AltogenBiosystems是一家生物技术公司,开发和制造用于生命科学研究、药物发现和开发的转染试剂盒。Altogen®体内转染试剂可有效地将生物分子导入靶组织。细胞转染试剂盒针对特定的癌细胞系和原代细胞进行了优化。通过先进的试剂配方和优化的转染方案实现货物分子(DNA、RNA、蛋白质)的高效传递。AltogenBiosystems利用高分子化学、分子和细胞生物学的专业知识,开发了新的体内外给药技术。转染是将外源分子导入培养细胞中,常用于研究基因功能、基因表达调控、生化定位和蛋白质生产。不幸的是,由于细胞毒性和转染效率的差异很大,并且取决于所使用的试剂、方案和细胞类型,因此没有一种单一的传递方法或转染试剂可应用于所有类型的细胞。AltogenBiosystems为120多个癌细胞系和原代细胞类型提供优化的转染试剂盒和电穿孔产品。体内转染试剂可实现组织靶向给药。Altogen的转染试剂盒包括用于体外(癌细胞系)和体内(用于动物研究的组织靶向试剂)转染的转染增强剂试剂和转染复合物冷凝器。Altogen实验室提供符合GLP的实验室合同研究服务。我们的生物CRO服务包括异种移植物的疗效、IND应用的pharm/tox研究和安全性测试、分析开发(ELISA、IC-50、qPCR)、90多个异种移植物动物模型、RNAi和基因沉默服务。Altogen的细胞培养科学家通过在28天内培育出稳定的细胞系,将选择的细胞系转化为稳定表达感兴趣的基因。
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转染技术的要点及转染试剂正确选择
转染技术是指将外源分子如DNA,RNA等导入真核细胞的技术,它是研究基因表达调控,突变分析等的常规工具。随着功能研究的兴起,其应用越来越广泛。以下就向大家介绍一些转染的技术要点及市面上主要的转染试剂类型的选择。
常规转染技术可分为两大类,一类是瞬时转染,一类是稳定转染(永久转染)。前者外源DNA/RNA不整合到宿主染色体中,因此一个宿主细胞中可存在多个拷贝数,产生高水平的表达,但通常只持续几天,多用于启动子和其它调控元件的分析。一般来说,超螺旋质粒DNA转染效率较高,在转染后24-96小时内(依赖于各种不同的构建)分析结果,常常用到一些报告系统如荧光蛋白,β半乳糖苷酶等来帮助检测。后者也称稳定转染,外源DNA既可以整合到宿主染色体中,也可能作为一种游离体(episome)存在。尽管线性DNA比超螺旋DNA转入量低但整合率高。外源DNA整合到染色体中概率很小,大约1/104转染细胞能整合,通常需要通过一些选择性标记,如来氨丙基转移酶(APH;新霉素抗性基因),潮霉素B磷酸转移酶(HPH),胸苷激酶(TK)等反复筛选,得到稳定转染的同源细胞系。
转染效率收多种因素影响,主要因素有下面几个:
1.细胞培养物
健康的细胞培养物是成功转染的基础。不同细胞有不同的培养基,血清和添加物。低的细胞代数(<50)能确保基因型不变。高的转染效率需要一定的细胞密度,一般的转染试剂都会有专门的说明。推荐在转染前24小时分细胞,这将提供正常细胞代谢,增加对外源DNA摄入的可能。一定要避免细菌,支原体或真菌的污染。
2.血清
大多数培养基在使用前需要加血清。胎牛血清(FCS)经常用到,便宜一点的有马或牛血清。通常的,血清是一种包含生长因子及其它辅助因子的不确切成分的添加物,对不同细胞的生长作用有很大的差别。血清质量的变化直接影响转染效率。因此在转染前建议先测(转右)试出对细胞生长良好的血清批号,转染时用同一批号的血清,并同时做负对照(不加转染试剂及外源DNA)以测试细胞生长是否正常。有些转染技术如脂质体转染在有血清存在情况下效率很低,因此在转染前要除血清。但有些对此敏感的细胞如原代细胞会受到损伤,甚至死亡导致转染效率极低。
3.载体构建
转染载体的构建(病毒载体,质粒DNA,RNA,PCR产物,寡核苷酸等)也影响转染结果。病毒载体对特定宿主细胞感染效率较高,但不同病毒载体有其特定的宿主,有的还要求特定的细胞周期,如逆转录病毒需侵染分裂期的宿主细胞,此外还需考虑一些安全问题(如基因污染)。除载体构建外,载体的形态及大小对转染效率也有不同的影响,如前面提到的超螺旋及线性DNA对瞬时和稳定转染的影响。如果基因产物对细胞有毒性作用,转染也很难进行,因此选择组成或可调控,强度合适的启动子也很重要,同时做空载体及其它基因的相同载体构建的转染正对照可排除毒性影响的干扰。
4.DNA质量
DNA质量对转染效率影响非常大。一般的转染技术(如脂质体等)基于电荷吸引原理,如果DNA不纯,如带少量的盐离子,蛋白,代谢物污染都会显著影响转染复合物的有效形成及转染的进行。核酸纯化世界第一品牌德国Qiagen公司提供的超纯质粒抽提试剂盒,能达到两倍2xCsCl梯度离心以上的纯度效果,使您不必为DNA质量操心。此外,对一些内毒素敏感的细胞(如原代细胞,悬浮细胞和造血细胞),QIAGEN还提供可去除内毒素污染的质粒抽提试剂盒,在质粒抽提过程中有效去除脂多糖分子,保证理想的转染效果。
5.转染技术
转染技术的选择对转染结果影响也很大,许多转染方法需要优化DNA与转染试剂比例,细胞数量,培养及检测时间等。一些传统的转染技术,如DEAE右旋糖苷法,磷酸钙法,电穿孔法,脂质体法各有利弊,其主要原理及应用特点见下:
转染方法原理应用特点
DEAE-右旋糖苷法
带正电的DEAE-右旋糖苷与核酸带负电的磷酸骨架相互作用形成的复合物被细胞内吞瞬时性转染相对简便、结果可重复但对细胞有一定的毒副作用,转染时需除血清
磷酸钙法
磷酸钙DNA复合物吸附细胞膜被细胞内吞稳定转染,瞬时性转染不适用于原代细胞,操作简便但重复性差,有些细胞不适用。
电穿孔法
高脉冲电压破坏细胞膜电位,DNA通过膜上形成的小孔导入稳定转染,瞬时性转染,所有细胞适用性广但细胞致死率高,DNA和细胞用量大,需根据不同细胞类型优化电穿孔实验条件
阳离子性的脂质体法
带正电的脂质体与核酸带负电的磷酸基团形成复合物被细胞内吞稳定转染
瞬时性转染,所有细胞适用性广,转染效率高,重复性好但转染时需除血清
转染效果随细胞类型变化大
病毒介导法
逆转录病毒通过侵染宿主细胞将外源基因整合到染色体中稳定转染,特定宿主细胞可用于难转染的细胞、原代细胞,体内细胞等,但携带基因不能太大
细胞需处分裂期,需考虑安全因素,腺病毒瞬时转染,特定宿主细胞可用于难转染的细胞,需考虑安全因素。
Biolistic颗粒传递法
将DNA用显微重金属颗粒沉淀,再将包被好的颗粒用弹道装置投射入细胞,DNA在胞内逐步释放,表达瞬时性转染可用于:人的表皮细胞,纤维原细胞,淋巴细胞系以及原代细胞
显微注射法
用显微操作将DNA直接注入靶细胞核稳定转染,瞬时性转染转染细胞数有限,多用于工程改造或转基因动物的胚胎细胞。
想用RNAiMAX或者lipo2000转染siRNA,但是看了下转染试剂的说明书和锐博的siRNA说明书,觉得分别对siRNA的用量描述差别挺大的呀,不知道到底该参考哪个呢。
以24孔板为例在siRNA说明书中写到,siRNA终浓度是50nM的话,加入浓度为20μM的siRNA1.25ul,每孔体积是500ul,那这样的话,每孔最终siRNA的量是25pmol。
但是在RNAiMAX或者是lipo2000说明书中,一个写的每孔siRNA用量是5pmol,一个是500ng,这与siRNA厂家所提供的量相差也太多了吧。
到底该看哪一个呢。
ps.一旦siRNA的量和体积确定下来之后,转染试剂的量和siRNA1:1的加就可以了吗?
请各位大神解答。
1.siRNA说明书中的用量,红线圈出
2.RNAIMAX说明书中siRNA的用量。
3.lipo2000说明书中siRNA用量
DXY721认为:
悬浮细胞和贴壁细胞在转染过程中差别不大,主要差别在于转染后的筛选,当然如果你做的是瞬时转染就不存在筛选的问题了。
其实转染的过程很简单,问题是能不能转的进去的,转染率能有多少,转进去是否可以稳定表达目的蛋白等等。
我们也是用脂质体做悬浮细胞的转染,说明书上都有具体的操作过程,将脂质体和目的基因按比例混合,然后加到细胞悬液里就OK了,说的简单,实际上还是有一些细节要注意的,比如脂质体和目的基因混合的比例,转染的细胞数,细胞的代数,细胞的状态,有的还要求在转染的前一天传代一次,不过不要怕,这些在脂质体说明书上都有明确的说明,按照说明书做就可以了。
jinghuanlv认为:
悬浮细胞和贴壁细胞转染还是有很大不同的。
脂质体转染的原理基于电荷吸引原理,先形成脂质体-DNA复合物,散布在细胞周围,然后通过细胞的内吞作用,将目的基因导入细胞内,而脂质体复合物与贴壁细胞的接触机会比悬浮细胞高出很多倍,所以,脂质体转染时悬浮细胞的转染效率要明显低于贴壁细胞。
我们实验室转染悬浮细胞是用的电穿孔法,目前为止,悬浮细胞转染的最好方法还是电转,我们实验室用的电转仪是Bio-Rad的,使用条件是电压250V,电容975uF,效果不错,不妨一用。
如题,PolyplusTransfection转染试剂在中国区的代理商有哪些?求推荐1-2个靠谱的,谢谢!
而且直接使用自制的PEI非常便宜,在293上远比商品化的脂质体要好。
另外,如果你们实验室确实钱多,不怕花钱,建议你取用Promega的FugenHD,那个转染效率比脂质体更好,而且毒性小,至于价格。。。。。。。。。。。也更高。。。。。。。。。
另外,你说漂浮的细胞有表到GFP,那个不一定是真的GFP,很多时候

