Analysis of the chicken genome for genes encoding embryonic stem cell indicators.

摘要

The development of efficient methods for establishing germline competent chicken embryonic stem (ES) cell lines has proved elusive. In the mouse embryo, expression of oct 3/4 is limited to pluripotent cells and primordial germ cells; regulatory sequences of this gene have been used to derive germline competent mouse ES cell lines by the continuous ablation of differentiated cells in culture using drug selection. To apply this technique to chickens several strategies were employed to analyze the chicken genome for oct 3/4, a member of the highly conserved POU gene family. PCR and Southern hybridization experiments with primers and probes based on mouse oct 3/4 sequences indicated that oct 3/4-like sequences are not present in the chicken genome. Also, analysis of mRNA from Stage 14 and 20 (H&H) chick embryos by reverse transcription PCR and the screening of a Stage 20 (H&H) chick embryo cDNA library with mouse oct 3 /4-based primers and probes indicated that oct 3/4-like sequences are not expressed in the early chick embryo. The apparent absence of oct 3/ 4 in chickens, despite the conservation of the gene in mammals and urodeles, is discussed in terms of possible implications on the mode of chicken PGC formation in relation to that in other vertebrates.; In the apparent absence of a chicken oct 3/ 4 equivalent, the use in this study of conserved mouse oct 3/4 primers resulted in the amplification of the chicken skn1/epoc1/oct 11 gene, which is a marker of terminally differentiating keratinocytes and another member of the POU gene family. The reproducible amplification by interspecific probes of one POU gene family member during the search for another has been reported several times by other groups and indicates the care that must be taken in analyzing this complex family.; Chicken stathmin, which encodes another suggested indicator of mouse pluripotent cells, was isolated and characterized in this study. Similarities in the coding sequences and expression of the chicken and mouse stathmin genes at certain stages of development suggest that their products are functional homologues, thus substantiating the argument for further investigations into the potential use of regulatory regions of the stathmin gene (as an alternative to those of oct 3/ 4) in the proposed system for the establishment of long term cultures of germline competent chicken ES cells.