The video demonstrates a procedure for injecting a plasmid mix into the ventricle of an anesthetized mouse pup. It outlines steps to locate the injection site, including identifying cranial sutures and using a glass pipette for precise delivery, emphasizing technique and accuracy during the injection process.

microinjection of dna plasmid mix into the mouse brain ventricle

エレクトロポレーションに基づくプラスミドDNAの導入によるin vivo脳腫瘍モデリング

Murine brain tumor models are crucial for understanding the mechanisms of brain tumor formation and treatment. Current models typically include rapidly produced subcutaneous or orthotopic transplantations of commonly used tumor cell lines, based on a limited number of driver mutations or patient-derived xenograft models, using immunodeficient mice that hinder proper immunotherapy studies1,2,3,4. Additionally, these preclinical results can lead to false positives, in that such models can exhibit dramatic, oftentimes curative effects in response to therapy, but this does not translate to the clinic2,5,6,7. Having the ability to rapidly produce genetically engineered preclinical mouse models that are more reflective of patient mutation signatures is imperative for improving the validity of preclinical results.
Electroporation (EP)-based delivery of DNA plasmids to induce both loss of function (LOF) and gain of function (GOF) mutations allows for the generation of such models. We developed a method for an even more precise representation of GOF driver mutations called mosaic analysis with dual-recombinase-mediated cassette exchange, or MADR8. This method allows for the expression of a gene (or genes) of interest in a controlled, locus-specific manner in somatic cells8. In combination with other molecular tools, such as clustered regularly interspaced short palindromic repeats (CRISPR), different patient mutations can be combined to develop mouse brain tumor models. This method has been used for different pediatric brain tumors, including gliomas and ependymomas8, as well as adult brain tumor models, such as glioblastoma (GBM).
While the EP method of tumor modeling is not as common as a transplant, the following demonstrates heretofore the ease and high reproducibility of this modeling system. mTmG mice are used for the insertion of the MADR-plasmid DNA8,9. This system allows for the recombination of loxP&#160;and Flp recombinase target (FRT) sites located at the Rosa26 locus for subsequent insertion of the donor DNA plasmid (i.e., GOF gene of interest)8,9. The following protocol demonstrates the straightforwardness of this method after diligent practice, and the ability to develop mouse brain tumor models in an autochthonous, consistent manner.

著者スポットライト:エレクトロポレーションベースのプラスミドDNA送達を用いた生体内での脳腫瘍のモデリング:患者の突然変異シグネチャーを表すプラスミドDNAの送達  

患者の突然変異シグネチャーを表すプラスミドDNAのエレクトロポレーションベースの送達を使用した in vivo での脳腫瘍のモデリング

Watch this Scientific Journal Video about Microinjection of DNA Plasmid Mix into the Mouse Brain Ventricle at JoVE.com

Microinjection of DNA Plasmid Mix into the Mouse Brain Ventricle  

マウス脳室へのDNAプラスミドミックスのマイクロインジェクション

まず、麻酔をかけたマウスの子犬を腹側の位置でテーブルの上に置きます。子犬の後頭部を少し引いて、頭蓋骨の縫合糸が皮膚を通して見えるようにします。頭蓋骨のラムダを見つけます。心室または注射部位は、ラムダと眼の中間に表示されます。垂直な角度で、ガラスピペットチップで皮膚を突き刺すと、初期抵抗とわずかな凹面のくぼみが観察されます。穴を開けたら、チップを所定の位置に保持し、マイクロインジェクターのフットペダルを1回踏んで、1マイクロリットルのプラスミド混合物を注入します。

microinjection-of-dna-plasmid-mix-into-the-mouse-brain-ventricle

Research

JoVE Journal

Cancer Research

Microinjection of DNA Plasmid Mix into the Mouse Brain Ventricle

185 ビュー. まず、麻酔をかけたマウスの子犬を腹側の位置でテーブルの上に置きます。子犬の後頭部を少し引いて、頭蓋骨の縫合糸が皮膚を通して見えるようにします。頭蓋骨のラムダを見つけます。心室または注射部位は、ラムダと眼の中間に表示されます。垂直な角度で、ガラスピペットチップで皮膚を突き刺すと、初期抵抗とわずかな凹面のくぼみが観察されます。穴?...

ビデオ: Microinjection of DNA Plasmid Mix into the Mouse Brain Ventricle  

Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.

Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model.

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in ...