SummaryThe bacteriophage P1 Cre-lox site-specific recombination system has been used to integrate DNA specifically at Iox sites previously placed in the tobacco genome. As integrated molecules flanked by wild-type Iox sites can readily excise in the presence of Cre recombinase, screening for mutant Iox sites that can resist excisional recombination was performed. In gene integration experiments, wild-type and mutant Iox sites were used in conjunction with two strategies for abolishing post-integration Cre activity: (i) promoter displacement of a cre-expression construct present in the target genome; and (ii) transient expression of cre. When the promoter displacement strategy was used, integrant plants were recovered after transformation with constructs containing mutant Iox sequences, but not with constructs containing wild-type Iox sites. When cre was transiently expressed, integrant plants were obtained after transformation with either mutant or wild-type Iox sites. DNA rearrangements at the target locus were less frequent when mutant Iox sites were used. DNA integration at the genomic Iox site was usually without additional insertions in the genome. Thus, the Cre-lox site-spesific recombination system is useful for the single-copy integration of DNA into a chromosomal Iox site.

In an effort to control the variability of transgene expression in plants, we used Cre-lox mediated recombination to insert a gus reporter gene precisely and reproducibly into different target loci. Each integrant line chosen for analysis harbors a single copy of the transgene at the designated target site. At any given target site, nearly half of the insertions give a full spatial pattern of transgene expression. The absolute level of expression, however, showed target site dependency that varied up to 10-fold. This substantiates the view that the chromosome position can affect the level of gene expression. An unexpected finding was that nearly half of the insertions at any given target site failed to give a full spatial pattern of transgene expression. These partial patterns of expression appear to be attributable to gene silencing, as low gus expression correlates with DNA methylation and low transcription. The methylation is specific for the newly integrated DNA. Methylation changes are not found outside of the newly inserted DNA. Both the full and the partial expression states are meiotically heritable. The silencing of the introduced transgenes may be a stochastic event that occurs during transformation.

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In situ hybridization of mouse embryo whole mounts and sagittal sections revealed a tissue- and stage-specific expression pattern of the transcriptionally regulated serum and glucocorticoid-inducible protein kinase (sgk) during embryogenesis. Sgk expression is first observed at embryonic day 8.5 (E8.5) in the decidua and yolk sac, and then during developmental stages E9.5 through E12.5 this kinase is highly localized in the heart chamber, otic vesicle, blood vessels surrounding the somites, and lung buds. At the later stages of mouse embryogenesis, E13.5 through E16.5, sgk expression becomes highly concentrated in brain (choroid plexus), distal epithelium and the terminal bronchi/bronchioles, adrenal gland, liver, thymus and intestines, remains high in heart tissue, and is expressed at a low level in the other embryonic tissues.