Highly effective ex vivo gene manipulation to study kidney development using self-complementary adenoassociated viruses.

Tie-Lin Chen,Yun-Hong Liu,Hong-Lian Wang,Rui-Zhi Tan,Xiao-Yan Lv,Yin Fang,Qin Zhou,Pu-Hui Zhou

doi:10.1155/2014/682189

Abstract

Background. Ex vivo culture of intact embryonic kidney has become a powerful system for studying renal development. However, few methods have been available for gene manipulation and have impeded the identification and investigation of genes in this developmental process. Results. Here we systemically compared eight different serotypes of pseudotyped self-complementary adenoassociated viruses (scAAVs) transduction in cultured embryonic kidney with a modified culture procedure. We demonstrated that scAAV was highly effective in delivering genes into and expressing in compacted tissues. scAAV serotypes 2 and 8 exhibited higher efficiency of transduction compared to others. Expression kinetics assay revealed that scAAV can be used for gene manipulation at the study of UB branching and nephrogenesis. Repressing WT1 in cultured kidney using shRNA impairs tubule formation. We for the first time employed and validated scAAV as a gene delivery tool in cultured kidney. Conclusions. These findings are expected to expedite the use of the ex vivo embryonic kidney cultures for kidney development research. For other ex vivo cultured organ models, scAAV could also be a promising tool for organogenesis study.

Highlights

Ex vivo intact organ cultures have become an excellent model for analysing normal and impaired organogenesis
Our results show that self-complementary adenoassociated viruses (scAAVs) is a highly effective tool for delivering gene into ex vivo cultured intact embryonic kidneys
Lentivirus and adenovirus were widely employed for in vivo and in vitro application, like siRNA transfection, they are unevenly distributed and appear to be excluded from the cap mesenchyme (Figure S1), which is in line with published papers [11, 14]. scAAV is a promising vector which gained much attention for gene transfer and gene therapy in the last decades [24]

Summary

Background

Ex vivo culture of intact embryonic kidney has become a powerful system for studying renal development. We systemically compared eight different serotypes of pseudotyped self-complementary adenoassociated viruses (scAAVs) transduction in cultured embryonic kidney with a modified culture procedure. We demonstrated that scAAV was highly effective in delivering genes into and expressing in compacted tissues. Expression kinetics assay revealed that scAAV can be used for gene manipulation at the study of UB branching and nephrogenesis. We for the first time employed and validated scAAV as a gene delivery tool in cultured kidney. These findings are expected to expedite the use of the ex vivo embryonic kidney cultures for kidney development research. For other ex vivo cultured organ models, scAAV could be a promising tool for organogenesis study

Introduction

Materials and Methods

Results

Discussion