Name: Microinjection of DNA Plasmid Mix into the Mouse Brain Ventricle
Uploaded: 2023-06-23T13:20:00
Description: Watch this Scientific Journal Video about Microinjection of DNA Plasmid Mix into the Mouse Brain Ventricle at JoVE.com

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

Modellazione in vivo di tumori cerebrali tramite somministrazione basata su elettroporazione di DNA plasmidico

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.

Autore in primo piano: Modellazione dei tumori cerebrali in vivo utilizzando la somministrazione basata sull'elettroporazione di DNA plasmidico che rappresenta le firme delle mutazioni del paziente  

Modellazione di tumori cerebrali in vivo utilizzando la somministrazione basata sull'elettroporazione di DNA plasmidico che rappresenta le firme di mutazione del paziente

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  

Microiniezione di mix di plasmidi di DNA nel ventricolo cerebrale del topo

Per iniziare, posiziona il cucciolo di topo anestetizzato sul tavolo in posizione ventrale. Tira leggermente la parte posteriore della testa del cucciolo per visualizzare le suture craniche del cranio attraverso la pelle. Trova lambda sul cranio.Il ventricolo o il sito di iniezione saranno visibili a metà strada tra lambda e l'occhio. Ad angolo perpendicolare, forare la pelle con la punta di una pipetta di vetro, si osserverà una resistenza iniziale e una leggera rientranza concava. Una volta forato, tenere la punta in posizione e premere una volta il pedale del microiniettore per iniettare un microlitro della miscela plasmidico.

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

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 ...