Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction

Wily G. Ruiz; Dennis R. Clayton; Marianela G. Dalghi; Nicolas Montalbetti; Marcelo D. Carattino; Gerard Apodaca

doi:10.3791/64584

아데노바이러스 매개 형질도입을 이용한 천연 방광 요로상피에서의 형질전환 유전자의 발현

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Biology

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JoVE Journal Biology

Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction

아데노바이러스 매개 형질도입을 이용한 천연 방광 요로상피에서의 형질전환 유전자의 발현

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October 6, 2022

DOI: 10.3791/64584-v

Wily G. Ruiz¹, Dennis R. Clayton¹, Marianela G. Dalghi¹, Nicolas Montalbetti¹, Marcelo D. Carattino¹, Gerard Apodaca¹

¹Renal-Electrolyte Division, Department of Medicine,University of Pittsburgh School of Medicine

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Overview

This study presents a protocol for the efficient transduction of native rodent urothelium using recombinant adenoviruses, enabling the expression of cDNAs and siRNAs. Key outcomes include achieving a transduction efficiency of nearly 95% in urothelial cells, facilitating research on urothelial biology and related diseases.

Key Study Components

Research Area

Urothelial biology
Gene expression regulation
Transduction techniques

Background

Importance of urothelium in bladder function
Challenges in studying gene expression in vivo
Need for effective transduction methods

Methods Used

Use of recombinant adenovirus for gene delivery
Mouse and rat models
Western blot and immunofluorescence assays for analysis

Main Results

Demonstrated high efficiency of transgene expression in urothelial cells
Confirmed expression of specific proteins, such as Claudin-2 and human growth hormone
Established that the adenovirus specifically transduces urothelium without affecting adjacent tissues

Conclusions

This protocol allows for in-depth studies of gene function in the urothelium
Offers insights into conditions affecting bladder biology, contributing to broader biological research

Frequently Asked Questions

What is the primary advantage of this method?

The method allows for high-efficiency expression of transgenes in a native biological context, which is critical for studying urothelial function.

Is the catheterization process difficult?

While it is considered the most challenging aspect, it becomes easier with practice and proper technique.

What types of assays were used to validate the findings?

Western blot and immunofluorescence assays were used to confirm protein expression in transduced urothelium.

How does this technique impact bladder disease research?

It enables researchers to explore the effects of gene overexpression or underexpression on bladder function and disease mechanisms.

Can this method be applied to other types of cells?

While the protocol is focused on urothelial cells, the underlying principles may be adapted for other tissue types.

What safety measures are necessary during the procedure?

Proper anesthetic management and biohazard waste disposal protocols must be followed to ensure animal and operator safety.

다량의 재조합 아데노바이러스의 생성에 대한 방법이 기재되어 있으며, 이는 이식유전자의 발현 또는 내인성 유전자 산물의 하향조절을 허용하는 천연 설치류 요로상피를 형질도입하는 데 사용될 수 있습니다.

이 프로토콜은 조사자가 천연 방광 요로상피에서 CDNA 또는 SIRNA를 발현할 수 있도록 하는 방법을 설명합니다. 이 기술의 주요 장점은 상대적으로 단순성, 요로상피에서 cDNA, siRNA를 고효율로 비교적 짧은 시간 내에 발현하는 능력입니다. 이 기술의 가장 어려운 부분은 카테터 삽입입니다.

그러나 전반적인 기술은 한 번 배우면 비교적 쉽게 마스터할 수 있습니다. 절차를 시연하는 것은 내 실험실의 연구 전문가 IV인 Giovanni Ruiz가 될 것입니다. 시작하려면 마취 된 마우스를 코 콘 옆의 가열 패드에 놓습니다.

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