Confirming Viral Transduction in Recellularized Rat Liver Using Immunohistochemical Staining

Abstract

Source: Hiller, T. et. al. Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2. J. Vis. Exp. (2016)

This video demonstrates immunohistochemical staining to evaluate viral gene transfer in a recellularized rat liver model. Reporter expression, confirmed by antibody detection and co-localization with nuclear staining, indicates successful viral transduction in the cells.

Protocol

1. Transduction of the Recellularized Rat Liver

1. Large-Scale Production of viral vector derived from adeno-associated viruses (AAV vector)
   1. Produce, purify, and quantify AAV vectors:
      1. Briefly, produce AAV vectors in roller bottles and purify them by iodixanol gradient centrifugation. Remove residual iodixanol by filtration over PD10 gel filtration columns. Determine the AAV vector concentration by quantitative polymerase chain reaction (qPCR) using genomic AAV DNA as a standard.
         NOTE: The self-complementary, pseudotyped AAV2/6 vector used in the present study enco....

Results

Figure 1. Map of the self-complementary, pseudotyped AAV2/6 vector used in the present study. The genome consists of the inverted terminal repeats (ITR) of AAV2 and encodes EmGFP under control of the CMV promoter as well as an shRNA under control of a U6.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tissue-Tek O.C.T.	Weckert-Labortechnik	600001
Cryomold 15 x 15 x 5 mm	Sakura	4566
Incubator	Fraunhofer	/