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

通过基于电穿孔的质粒 DNA 递送进行体内脑肿瘤建模

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.

作者聚焦：使用基于电穿孔的质粒 DNA 递送代表患者突变特征的体内脑肿瘤建模  

使用基于电穿孔的质粒DNA递送对 体内 脑肿瘤进行建模，以代表患者突变特征

The technique focuses on modeling patient mutation profiles of glioblastoma in the immunocompetent mouse model and how these different mutations are influencing the tumor microenvironment and response to treatment. Genomic and single cell sequencing identify that the genetic driver mutations of glioblastoma are associated with the aggressiveness of cancer and components of the tumor microenvironment such as the immune cell population. Glioblastoma mouse models in preclinical trials show great success with immunotherapy leading to curative results and no sign of tumor growth after treatment.However, these effects are not reflected in patients for outcome or survival. This protocol recapitulates patient tumor mutation profiles of glioblastoma in the immunocompetent mouse model, allowing gradual autochthonous tumor growth and better prediction of treatment efficacy, especially with immunotherapy. This modeling system integrates DNA plasmids into the genome through electroporation of immunocompetent mice.It avoids the use of viruses and transplanting thousands of tumor cells, both of which can have their own immune effects, confounding downstream results. Utilizing an immunocompetent mouse model that recapitulates patient mutation profiles allows for a more accurate assessment of treatment outcomes, preventing the laborious task of taking false positive results to the clinic.

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  

将DNA质粒混合物显微注射到小鼠脑室中

首先，将麻醉的小鼠幼崽放在腹侧位置的桌子上。轻轻拉动幼犬的后脑勺，以观察颅骨缝合线穿过皮肤。在头骨上找到lambda。心室或注射部位将在 lambda 和眼睛之间可见。以垂直角度，用玻璃移液器吸头刺穿皮肤，将观察到初始阻力和轻微的凹陷。刺穿后，将尖端固定到位并按下微注射器脚踏板一次以注射一微升质粒混合物。

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 次观看.  Cedars-Sinai Medical Center. 首先，将麻醉的小鼠幼崽放在腹侧位置的桌子上。轻轻拉动幼犬的后脑勺，以观察颅骨缝合线穿过皮肤。在头骨上找到lambda。心室或注射部位将在 lambda 和眼睛之间可见。以垂直角度，用玻璃移液器吸头刺穿皮肤，将观察到初始阻力和轻微的凹陷。刺穿后，将尖端固定到位并按下微注射器脚踏板一次以注射一微升质粒混合物。

视频: 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 ...

科研

癌症研究

To begin, place the anesthetized mouse pup on the table in the ventral position. Pull the back of the pup's head slightly to visualize the cranial skull sutures through the skin. Find lambda on the skull.The ventricle or injection site will be visible midway between lambda and the eye. At a perpendicular angle, pierce the skin with a glass pipette tip, an initial resistance and slight concave indentation will be observed. Once pierced, hold the tip in place and press the micro injector foot pedal once to inject one microliter of the plasmid mix.

Electroporation-Based DNA Plasmids Delivery to Induce In Vivo Mutations in the Mouse

Begin electroporation-based DNA plasma delivery by placing a square piece of paraffin film on the table and squeezing the electrode gel on top of the film. Then apply a generous amount of the electrode gel on each paddle to cover the electrode paddles completely. Next, hold the pup micro-injected with the plasmid DNA mix in the non-dominant hand, keeping the fingers behind the head.To position the pup's head for electroporation, gently slide a finger on the next dorsal side, posterior to the head, and position the positive electrode outside the head where the ventricle is targeted. Place the electrode over the pup's eye and lightly squeeze the electrode paddles on the rostral part of the pup's head. Press the electrode foot pedal to start the electroporation.With each pulse, rotate the electrode paddles slightly, in a counterclockwise direction, so the positive paddle moves towards the top of the head. After the final pulse, remove the paddles and place them aside. Mice were moribund by five months post-electroporation, with full tumor growth detected through the reporter protein and histological analysis.