Real-time visualization of human prolactin alternate promoter usage in vivo using a double-transgenic rat model.

We have generated a humanized double-reporter transgenic rat for whole-body in vivo imaging of endocrine gene expression, using the human prolactin (PRL) gene locus as a physiologically important endocrine model system. The approach combines the advantages of bacterial artificial chromosome recombineering to report appropriate regulation of gene expression by distant elements, with double reporter activity for the study of highly dynamic promoter regulation in vivo and ex vivo. We show first that this rat transgenic model allows quantitative in vivo imaging of gene expression in the pituitary gland, allowing the study of pulsatile dynamic activity of the PRL promoter in normal endocrine cells in different physiological states.

Cryptic loxP sites in mammalian genomes: genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques.

Cre is widely used for DNA tailoring and, in combination with recombineering techniques, to modify BAC/PAC sequences for generating transgenic animals. However, mammalian genomes contain recombinase recognition sites (cryptic loxP sites) that can promote illegitimate DNA recombination and damage when cells express the Cre recombinase gene. We have created a new bioinformatic tool, FuzznucComparator, which searches for cryptic loxP sites and we have applied it to the analysis of the whole mouse genome.

Granulation rescue and developmental marking of juxtaglomerular cells using "piggy-BAC" recombination of the mouse ren locus.

Mice lacking a functional Ren-1(d) gene exhibit a complete lack of renal juxtaglomerular cell granulation and atypical macula densa morphology. Transgenic mice carrying a 145-kilobase BAC clone encompassing the Ren-1(d) and Ren-2 loci were generated, characterized, and backcrossed with Ren-1(d-/-) mice. Homozygous Ren-1(d)-null mice expressing the BAC clone exhibited complete restoration of normal renal structure.

Efficient Cre-lox linearisation of BACs: applications to physical mapping and generation of transgenic animals.

Due to the size of BAC, PAC and P1 clones, it is often difficult to construct detailed restriction maps, with large number of restriction fragments leading to ambiguity of mapping data. We report the use of Cre recombinase to linearise and asymmetrically introduce label at the unique loxP site of large loxP-containing clones. Subsequent partial digestion allows the direct ordering of restriction fragments.

Identification of Grf1 on mouse chromosome 9 as an imprinted gene by RLGS-M.

Normal mammalian development requires a diploid combination of both haploid parental genomes. Uniparental disomy for certain segments of specific chromosomes results in aberrant development or prenatal lethality, indicating that the parental genomes have undergone modifications during gametogenesis. These modifications result in parent-of-origin specific expression for some genes, a phenomenon called genomic imprinting.