Lineage analysis of inner ear cells using genomic tags for clonal identification.
To understand the mechanisms of development of the inner ear, it is important to know the lineal relationships among the different cell types and the migrational boundaries of individual clones within the inner ear. This chapter details the basic methods for performing lineage analysis of the inner ear using replication-defective retroviral vectors in chicken embryos. Protocols are provided for generating avian retroviruses pseudotyped with vesicular stomatitis virus (VSV) envelopes to improve infectivity in early embryos. Moreover, we include the pioneering methods of the Cepko laboratory, whereby a library of DNA tags was developed to allow clonal relationships to be confirmed by PCR amplification and sequencing of the tag in dispersed clonal progeny. By varying the site and time of viral delivery, these methods are appropriate to study cell lineages in other tissues of the developing chicken.
Department of Biological Sciences, Purdue University, West Lafayette, IN, USA.
Takunori
T
Satoh
Donna M
DM
Fekete
D000818:Animals; D019070:Cell Lineage; D002642:Chick Embryo; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D007758:Ear, Inner / Q000166:cytology* / Q000196:embryology / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D008954:Models, Biological; D016133:Polymerase Chain Reaction / Q000379:methods; D012190:Retroviridae / Q000235:genetics; D016324:Sequence Tagged Sites