METHODS OF CONSTRUCTING A MODEL OF CELLULAR DEVELOPMENT AND DIFFERENTIATION USING HOMOZYGOUS STEM CELL SYSTEMS, METHODS OF ASSESSING AND CATALOGING PROTEINS EXPRESSED THEREIN, CDNA LIBRARIES GENERATED THEREFROM, AND MATERIALS AND METHODS USING SAME

摘要

The present invention relates to materials and methods for the quantitativeand qualitative analysis of gene expression during targeted differentiation ofisolated homozygous stem cells, and to materials and methods for accumulatingsequence tags sampled from a population of expressed genes. More particularly,the present invention relates to a method for constructing a model of cellulardevelopment and differentiation comprising the steps of: (1) producing ahomogenous population of homozygous stem (HS) cells; (2) directing thedifferentiation of the HS cells to arrive at desired mature (somatic) cells,group of cells or tissue; (3) periodically sampling HS cells undergoingdifferentiation to extract total cellular RNA produced at various samplingstages; (4) isolating mRNA from the total cellular RNA; and (5) constructing acDNA library representative of proteins produced and expressed at variousstages of differentiation, over the course of time. The present inventionfurther relates to sequencing and cataloguing isolated mRNAs and cDNAs atvarious stages of differentiation of HS cells, producing and cataloguingisolated polypeptides sequences encoded by same, and screening the cDNAlibraries. I vitro and in vivo cultures of homozygous stem cell are alsodescribed; such cultures find utility both as models of differentiation aswell as sources of differentiated tissues and organs.