, Volume 347, Issue 2, pp 357-367

Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer

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Abstract

The prospect of developing large animal models for the study of inherited diseases, such as cystic fibrosis (CF), through somatic cell nuclear transfer (SCNT) has opened up new opportunities for enhancing our understanding of disease pathology and for identifying new therapies. Thus, the development of species-specific in vitro cell systems that will provide broader insight into organ- and cell-type-specific functions relevant to the pathology of the disease is crucial. Studies have been undertaken to establish transformed rabbit airway epithelial cell lines that display differentiated features characteristic of the primary airway epithelium. This study describes the successful establishment and characterization of two SV40-transformed rabbit tracheal epithelial cell lines. These cell lines, 5RTEo- and 9RTEo-, express the CF transmembrane conductance regulator (CFTR) gene, retain epithelial-specific differentiated morphology and show CFTR-based cAMP-dependent Cl ion transport across the apical membrane of a confluent monolayer. Immunocytochemical analysis indicates the presence of airway cytokeratins and tight-junction proteins in the 9RTEo- cell line after multiple generations. However, the tight junctions appear to diminish in their efficacy in both cell lines after at  least 100 generations. Initial SCNT studies with the 9RTEo- cells have revealed that SV40-transformed rabbit airway epithelial donor cells can be used to generate blastocysts. These cell systems provide valuable models for studying the developmental and metabolic modulation of CFTR gene expression and rabbit airway epithelial cell biology.

We dedicate this manuscript to Dr. Xiangzhong Yang (deceased) whose enthusiasm and support was an inspiration during the work described in this manuscript.
This study was supported by NIH grant HL80814 and grants from the Cystic Fibrosis Foundation and from Pennsylvania Cystic Fibrosis. B.I. was also the recipient of a donation from the Folger Foundation. VRT-532, CFTR-inh172 and GlyH-101 were kindly provided to B.I. by the CFTR Compound Distribution Program of Cystic Fibrosis Foundation Therapeutics.