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We have devised a strategy (called recombinase-mediated genomic replacement, RMGR) to allow the replacement of large segments (>100 kb) of the mouse genome with the equivalent human syntenic region. The technique involves modifying a mouse ES cell chromosome and a human BAC by inserting heterotypic lox sites to flank the proposed exchange interval and then using Cre recombinase to achieve segmental exchange. We have demonstrated the feasibility of this approach by replacing the mouse alpha globin regulatory domain with the human syntenic region and generating homozygous mice that produce only human alpha globin chains. Furthermore, modified ES cells can be used iteratively for functional studies, and here, as an example, we have used RMGR to produce an accurate mouse model of human alpha thalassemia. RMGR has general applicability and will overcome limitations inherent in current transgenic technology when studying the expression of human genes and modeling human genetic diseases.

Lactase-phlorizin hydrolase (LPH), a major digestive enzyme in the small intestine of newborns, is synthesized as a highmolecular-mass precursor comprising four tandemly repeated domains. Proteolytic cleavage of the precursor liberates the pro segment (LPHα) corresponding to domains I and II and devoid of known enzymic function. The mature enzyme (LPHβ) comprises domains III and IV and is anchored in the brush border membrane via a C-terminal hydrophobic segment. To analyse the roles of the different domains of LPHα and LPHβ, and the interactions between them, we have engineered a series of modified derivatives of the rat LPH precursor. These were expressed in cultured cells under the control of a cytomegalovirus promoter. The results show that recombinant LPHβ harbouring both domains III and IV produces lactase activity. Neither domain III nor IV is alone sufficient to generate active enzyme, although the corresponding proteins are transport-competent.

hct-1 (hippocampal transcript) was detected in a differential screen of a rat hippocampal cDNA library. Expression of hct-1 was enriched in the formation but was also detected in rat liver and kidney, though at much lower levels; expression was barely detectable in testis, ovary, and adrenal. In liver, unlike brain, expression was sexually dimorphic; hepatic expression was greatly reduced in female rats. In mouse, brain expression was widespread, with the highest levels being detected in corpus callosum; only low levels were detected in liver. Sequence analysis of rat and mouse hct-1 cDNAs revealed extensive homologies with cytochrome P450s (CYPs), a diverse family of heme-binding monooxygenases that metabolize a range of substrates including steroids, fatty acids, and xenobiotics. Among the CYPs, hct-1 is most similar (39% at the amino acid sequence) to cholesterol 7␣-hydroxylase (CYP7) and contains a postulated steroidogenic domain present in other steroidmetabolizing CYPs but clearly represents a type of CYP not previously reported. Genomic Southern analysis suggests that a single gene corresponding to hct-1 is present in mouse, rat, and human. hct-1 is unusual in that, unlike all other CYPs described, the primary site of expression is in the brain. Similarity to CYP7 and other steroid-metabolizing CYPs may argue that hct-1 (CYP7B) plays a role in steroid metabolism in brain, notable because of the documented ability of brain-derived steroids (neurosteroids) to modulate cognitive function in vivo.Cytochromes P450, a diverse group of heme-containing monooxygenases (termed CYPs) 1 (for nomenclature see Nelson et al. (1)), catalyze a variety of oxidative conversions, notably of steroids but also of fatty acids and xenobiotics. Though most abundantly expressed in the testis, ovary, placenta, adrenal, and liver, the brain is a further site of CYP expression (2-6).Several CYP activities or mRNAs have been reported in the nervous system, predominantly of types metabolizing fatty acids and xenobiotics (subclasses CYP2C, -2D, -2E, and -4 (6, 7)). However, primary rat brain-derived glial cells can synthesize pregnenolone and progesterone in vitro (8). Mellon and Deschepper (9) provided molecular evidence for the presence, in brain, of key steroidogenic enzymes CYP11A1 (scc) and CYP11B1 (11␤) but failed to detect CYP17 (c17) or CYP11B2 (AS). Though CYP21A1 (c21) activity is reported to be present in brain (10) authentic CYP21A1 transcripts were not detected (11).Interest in brain steroid metabolism has been fueled by the finding that adrenal-and brain-derived steroids (neurosteroids) can modulate cognitive function and synaptic plasticity (reviewed in Refs. 12-17). For instance, pregnenolone and steroids derived from it are reported to have memory-enhancing effects in mice (18,19). However, the full spectrum of brain CYPs and the biological roles of their metabolites in vivo have not been established.To investigate such regulation of brain function our studies have focused on the hippocampus, a brain region important ...

The small GTPase Rab5 is a key regulator of endosome/phagosome maturation and in intravesicular infections marks a phagosome stage at which decisions over pathogen replication or destruction are integrated. It is currently unclear whether Leishmania‐infected phagosomes uniformly pass through a Rab5+ stage on their intracellular path to compartments with late endosomal/ early lysosomal characteristics. Differences in routes and final compartments could have consequences for accessibility to antileishmanial drugs. Here, we generated a unique gfp‐rab5 transgenic mouse model to visualize Rab5 recruitment to early parasite‐containing phagosomes in primary host cells. Using real‐time fluorescence imaging of phagosomes carrying Leishmania mexicana, we determined that parasite‐infested phagosomes follow a uniform sequence of transient Rab5 recruitment. Residence in Rab5+ compartments was much shorter compared with phagosomes harboring latex beads. Furthermore, a comparative analysis of parasite life‐cycle stages and mutants deficient in lpg1, the gene encoding the enzyme required for synthesis of the dominant surface lipophosphoglycan, indicated that parasite surface ligands and host cell receptors modulate pathogen residence times in Rab5+ phagosomes, but, as far as tested, had no significant effect on intracellular L. mexicana survival or replication.—Lippuner, C, Paape, D., Paterou, A., Brand, J., Richardson, M., Smith, A. J., Hoffmann, K., Brinkmann, V., Blackburn, C, Aebischer, T. Real‐time imaging of Leishmania mexicana‐infected early phagosomes: a study using primary macrophages generated from green fluorescent protein‐Rab5 transgenic mice. FASEB J. 23, 483–491 (2009)

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Chromosomal deletions are a common feature of epithelial tumours and when further de®ned by homozygous deletions, are often the location of tumour suppressor genes. Deletions within the short arm of chromosome 3 occur very frequently in human carcinomas: a minimal region of loss at 3p21.3 (the Luca) region has been de®ned by overlapping homozygous deletions in lung and breast cancer cell lines. Using a rapid strategy for Cre-loxP chromosome engineering, a deletion of approximately 370 kb was created in the mouse germline corresponding to the deleted region at 3p21.3. The deletion when homozygous is embryonic lethal. Heterozygotes develop normally despite being haplo-insucient for twelve genes including the candidate tumour suppressor gene Rassf1. Because damage to 3p21.3 often occurs very early in the sequence of genetic changes that lead to malignancy, particularly in lung and breast cancer, further genetic damage to these mice will provide the opportunity to model multi-step tumorigenesis of these tumours.

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