この製品は動物モデルへのプラスミドDNAの輸送用に設計されています。In Vivo DNA-Fectは市販のPEIベースのin vivo用試薬よりもトランスフェクション効率が高く、費用対効果が高い代替品となります。従来の試薬は、その毒性によって導入遺伝子の発現が制限されたり、投薬された箇所の周辺の生理反応が変化するなどしてしまうために、利用が限定されていました。In Vivo DNA-Fectはその生体適合性によって、細胞の生理機能の変化を最小化し、発現した導入遺伝子をより良く応答させます。
Rose et al., Biomaterials Science (2014) 2: 833-842.
Rose et al., Biomaterials (2012) 33: 3363-3374.
写真：ラットの皮下組織に2種のGFPプラスミドをそれぞれトランスフェクションした際に見られた、In Vivo DNA-Fectの典型的な性能。実験では、In Vivo DNA-Fectと分岐鎖PEI (PEI25; 25 kDa)を、それぞれの複合体をゼラチンスポンジに移植してから14日後に比較しています。導入遺伝子の発現は、移植片に蛍光顕微鏡を用いて組織学的に分析されました。
In Vivo RNA-Fect
この製品はマウス腫瘍モデルへのsiRNA輸送用に設計されています。特定箇所(例：腫瘍内)への直接投与にも、体系的(例：腹腔内投与、静脈内投与)にも使用できます。In Vivo RNA-Fectは、市販のPEIベースのin vivo用試薬よりも費用対効果が高い代替品となります。毒性が低いため、正常な動物モデルの健康に影響を与えることなく、核酸をより多く投与することが可能です。
One area of focus for RJH Biosciences is to implement RNA interference (RNAi) via delivery of short interfering RNA (siRNA). Our initial therapeutic application is blood cancers, while recognizing that the RNAi activity can be implemented in the treatment of a large range of human cancers and other diseases. Another focus is direct administration of plasmid DNA (pDNA) to express therapeutic proteins in situ, with applications in immunotherapy.
Why study blood cancers and immunotherapies with nucleic acid therapeutics?
There are three types of blood cancers: Leukemia, lymphoma, and myeloma. Leukemia is characterized by highly proliferating, abnormal white blood cells [1]. Lymphoma and myeloma are respectively cancers of the lymphatic system and plasma cells which greatly effect the immune system [2,3]. These three cancers are difficult to treat and the current treatments are limited in efficacy, especially at the end stage of the disease.
The use of nucleic acid-based therapeutics can eradicate these cancers in two primary ways, with RNAi technology and cell-based immunotherapy. The use of RNAi is being increasingly explored in the treatment of the blood cancers. Polynucleotides such as siRNA has aided the downregulation of oncogenes and can be designed to support specific abnormalities in individual patients, making it a ‘personal’ strategy with a universal technological design [4]. Due to siRNA’s potential in blood cancer therapies, we are currently focusing on siRNA therapeutics in our R&D projects, targeting disease-driving oncogenes and inducing apoptosis in the malignant cells.
Another strategy that is being explored for treating blood cancers is the use of immunotherapy. Immunotherapeutic strategies include the use of antibodies, stem cell transplants, cytokines, small molecules among others [5]. However, a more recent approach is genetic therapy by using engineered cells, also known as Cell Transfer Therapy. This method works by taking patients’ own immune cells such as but not limited to T-cells, B-cells, and NK cells. The immune cells genome is engineered to support various therapeutic strategies that may involve neoantigen expression and presentation on immune cell surface, and then are reintroduced into the host [5]. The modified cells are ultimately designed to target and remove the malignant cells. This strategy is highly advantageous as T-cells can ‘seek’ and destroy the malignant cells in the blood system. While this approach has been promising in blood cancers, it can be also used in other solid cancers. As the foundation of immunotherapy relies on nucleic acid introduction into patient cells, efficient delivery of nucleic acids is imperative for success. Our transfection reagents offer the best in class vehicles to undertake such a delivery.
Nanomedicine based on nucleic acid therapeutics is a large component to personalized cancer therapies and immunotherapies. The RJH Biosciences strives to provide quality transfection reagents, whether it involves the delivery of our own nucleic acid candidates or our customers’.
Jean, C. and Dick, J. (2005) Cancer stem cells: lessons from leukemia. Trends in cell biology. 15, 494-501. Woods, N. et al. (2006) Therapueti gene causing lymphoma. Nature. 440, 1123. Mahindra, A. et al. (2012) Latest advances and current challenges in the treatment of multiple myeloma. Nature Reviews Clinical Oncology. 9, 135-143. Uludağ, H. et al. (2016) Current attempts to implement siRNA-based RNAi in leukemia models. Drug Discovery Today. 21, 1412-1420. Zou, W. (2006) Regulatory T cells, tumour immunity and immunotherapy. Nature Reviews Immunology. 6, 295-307.
As the first step, we suggest consulting the ‘Transfection Reagent Selection Guide’ to find out if the cells of interest have been previously tested. If your cell type is not on the list, we suggest using the reagents optimized for similar cells (i.e., attachment-dependent/suspension or established cell line/primary cells), or contact us by e-mail for an informed suggestion.
We recommend small scale preliminary experiments to optimize the performance of our transfection reagents. As with all transfection reagents, the complex formation, cell seeding density, and culture and incubation conditions will affect the final performance. Please consult our technical sheet on optimizing transfection for this purpose (link).
Each product page has a copy their specific transfection protocol, they can be found here:
Our transfection reagents are compatible with a wide range of serum-free media, including DMEM, RPMI, MEM and others. Complexes are expected to be functional in serum-containing medium.
The recommended nucleic acid to transfection reagent ratio ranges from (w/w) 1:1 for relatively toxic reagents to 5-20:1 for biocompatible reagents. This ratio should be optimized for each application. For suggested ranges for each reagent, please consult the specific reagent manual.
No. It not necessary to remove the complexes from treated cells. Complexes can be left in culture with cells until end-point analysis.
Depending on the application, incubation times may vary from 2 hours to 24 hours. It may be possible to centrifuge the treated cells to accelerate the transfection process and minimize the complex incubation times.
Transfection efficiency can be determined by different approaches. Reporter genes, such as GFP or RFP, are convenient ways to assess transfection by using microscopy or flow cytometry techniques. Direct assessment of the induced gene product (e.g., by ELISA, western blot), or silenced gene expression (e.g., protein levels or mRNA levels by PCR) are important. One can also use functional outcomes as an indirect measure of transfection, although care must be paid to complicating factors in this case. In all studies, we recommend employing a control (i.e., non-active) agent similar in nature to the nucleic acid being investigated.
A methodical analysis of the factors contributing to transfection, as outlined in the ‘Technical Tips to Improve Transfection’, is a good place to start. Other resources to improve transfection efficiencies can be found in our ‘suggested reading’ tab. We are here to help as well, so do send us a message/e-mail to see how we can assist you.
All transfection reagents display a certain extent of cytotoxicity on cells, depending on the amount used. The key is to achieve transfection without disrupting the physiology of the cells significantly. One can minimize exposure time to transfection complexes, speed up the transfection process by centrifugation and optimize reagent/nucleic acid concentrations to eliminate unnecessary exposure. The purity of the nucleic acid is also important to eliminate unforeseen toxicities.
These are important criteria that affect the transfection efficiency. In general, transfection efficiency decreases with increased cell density and passage number, as cells settle in senescence. For other factors affecting transfection efficiency, please consult ‘Technical Tips to Improve Transfection’.
The recommend storage temperature is at 4 degC (short term) or -20 degC (long term). The reagents are designed to be stable for 1 year under these conditions.
We recommend to use 1:5 ratio of nucleic acid to transfection reagent, so that 1 mL vial (1 mg) is suitable for 200 transfections of 1 mg nucleic acid. However, optimal ratio may change depending on the application and cell type.
Yes. Our in vivo transfection reagents display broad activities so that they could be effective under in vitro conditions.
It is likely for our transfection reagents to work with different nucleic acids. This is not universally applicable, but most reagents seem to handle different nucleic acids. ALL-Fect transfection reagent can handle both DNA and RNA.
Our reagents are based on an optimal balance of cationic charge and hydrophobicity. They are polymeric in nature that interact with nucleic acids via multivalent interactions. These reagents provide effective condensation of anionic charge of nucleic acids, while displaying little toxicity on mammalian cells.
To indicate the source of the transfection reagent, you can state the reagent name and that it was obtained from RJH Biosciences Inc. (Edmonton, AB, Canada).
