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Immortalized keratinocytes can offer a low‐cost experimental platform for human skin research, with increased cell yield compared to primary cultures. However, the usefulness of these surrogate cell models is highly dependent on their ability to retain the phenotypic attributes of the parent cells. Keratins K14 and K5 are the hallmarks of undifferentiated, mitotically active basal keratinocytes. We observed occasional progressive loss of K14 expression in growing keratinocyte cell lines, with persistent retention of K5 and an epithelial phenotype, and investigated possible reasons for this. We show that K14 repression occurs by DNA promoter methylation of KRT14 gene and is compounded by histone deacetylation and by the presence of EGF. In vivo, keratinocytes shut down K14 synthesis as they commit to terminal differentiation and move from the basal to spinous layer, but by laser‐capture microdissection of human epidermis we could detect no evidence of increased selective KRT14 methylation in this normal process. Loss of K14 expression suggests that epidermal identity of cultured keratinocytes can be compromised in certain tissue culture situations, possibly due to the immortalization method and persistent EGF supplementation. [ABSTRACT FROM AUTHOR]

Research has been conducted on Escherichia coli insertion elements IS3, IS150 and IS186. The authors demonstrate that these elements can produce transpositionally active heat-to-tail dimers with the structure and transpositional activity similar to the dimers of IS2, IS21 and IS30.

A two-element Activator/Dissociation ( Ac/ Ds) gene trap system was successfully established in rice ( Oryza sativa ssp. japonica cv. Nipponbare) to generate a collection of stable, unlinked and single-copy Ds transposants. The germinal transposition frequency of Ds was estimated as an average of 51% by analyzing 4413 families. Study of Ds transposition pattern in siblings revealed that 79% had at least two different insertions, suggesting late transposition during rice development. Analysis of 2057 Ds flanking sequences showed that 88% of them were unique, whereas the rest within T-DNA. The insertions were distributed randomly throughout the genome; however, there was a bias toward chromosomes 4 and 7, which had two times as many insertions as that expected. A hot spot for Ds insertions was identified on chromosome 7 within a 40-kbp region. One-third of Ds flanking sequences was homologous to either proteins or rice expressed sequence tags (ESTs), confirming a preference for Ds transposition into coding regions. Analysis of 200 Ds lines on chromosome 1 revealed that 72% insertions were found in genic region. Anchoring of more than 800 insertions to yeast artificial chromosome (YAC)-based EST map showed that Ds transposes preferentially into regions rich in expressed sequences. High germinal transposition frequency and independent transpositions among siblings show that the efficiency of this system is suitable for large-scale transposon mutagenesis in rice. [ABSTRACT FROM AUTHOR]