The video demonstrates the electroporation technique for delivering DNA plasmids in mice. It outlines the procedure, from preparing electrodes with gel to positioning the pup's head for effective plasma delivery. The results reveal tumor growth by five months post-electroporation, confirmed through protein and histological analysis.

electroporation-based dna plasmids delivery to induce in vivo mutations in the mouse

플라스미드 DNA의 전기천공법 기반 전달을 통한 In vivo 뇌종양 모델링

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.

저자 스포트라이트: Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient mutation signatures  

Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures(환자 돌연변이 시그니처를 나타내는 플라스미드 DNA의 전기천공법 기반 전달을 사용한 생체 내 뇌종양 모델링)

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 Electroporation-Based DNA Plasmids Delivery to Induce In Vivo Mutations in the Mouse at JoVE.com

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

마우스에서 In Vivo 돌연변이를 유도하기 위한 전기천공법 기반 DNA 플라스미드 전달

정사각형의 파라핀 필름을 테이블 위에 놓고 필름 위에 전극 겔을 압착하여 전기천공법 기반 DNA 플라즈마 전달을 시작합니다. 그런 다음 전극 패들을 완전히 덮기 위해 각 패들에 전극 젤을 넉넉하게 바릅니다. 다음으로, 플라스미드 DNA 혼합물이 미세 주입된 강아지를 우세하지 않은 손에 잡고 손가락을 머리 뒤에 둡니다.전기 천공법을 위해 강아지의 머리를 배치하려면 머리 뒤쪽의 다음 등쪽에 손가락을 부드럽게 밀고 심실이 목표로 하는 머리 바깥쪽에 양극을 배치합니다. 전극을 강아지의 눈 위에 놓고 강아지 머리의 부분에 전극 패들을 가볍게 쥐어짜줍니다. 전극 풋 페달을 눌러 electroporation을 시작하십시오.각 펄스에서 전극 패들을 시계 반대 방향으로 약간 돌려 양극 패들이 머리 꼭대기로 이동하도록 합니다. 마지막 펄스 후 패들을 제거하고 따로 보관하십시오. 마우스는 전기천공법 후 5개월이 지났을 때 리포터 단백질과 조직학적 분석을 통해 전체 종양 성장이 감지되었습니다.

electroporation-based-dna-plasmids-delivery-to-induce-vivo-mutations

Research

JoVE Journal

Cancer Research

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

138 조회수.  Cedars-Sinai Medical Center. 정사각형의 파라핀 필름을 테이블 위에 놓고 필름 위에 전극 겔을 압착하여 전기천공법 기반 DNA 플라즈마 전달을 시작합니다. 그런 다음 전극 패들을 완전히 덮기 위해 각 패들에 전극 젤을 넉넉하게 바릅니다. 다음으로, 플라스미드 DNA 혼합물이 미세 주입된 강아지를 우세하지 않은 손에 잡고 손가락을 머리 뒤에 둡니다.전기 천공법을 위해 강아지의 머리를 배치하려?...

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

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 ...

연구

암 연구학

Microinjection of DNA Plasmid Mix into the Mouse Brain Ventricle

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.

DNA Plasmid Mix를 마우스 뇌실에 미세주입

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.