Abdulsalam Soofi

We report a transgenic mouse line that expresses Cre recombinase exclusively in podocytes. Twenty- four transgenic founders were generated in which Cre recombinase was placed under the regulation of a 2.5-kb fragment of the human NPHS2 promoter. Previously, this fragment was shown to drive beta-galactosidase (beta-gal) expression exclusively in podocytes of transgenic mice. For analysis, founder mice were bred with ROSA26 mice, a reporter line that expresses beta-gal in cells that undergo Cre recombination. Eight of 24 founder lines were found to express beta-gal exclusively in the kidney. Histological analysis of the kidneys showed that beta-gal expression was confined to podocytes. Cre recombination occurred during the capillary loop stage in glomerular development. No evidence for Cre recombination was detected in any of 14 other tissues examined.

Transgenic manipulation of the glomerular visceral epithelial cell offers a powerful approach for studying the biology of this morphologically complex cell type. It has been previously demonstrated that an 8.3-kb and a 5.4-kb fragment of the murine Nphs1 (nephrin) promoter-enhancer drives lacZ expression in podocytes, brain, and pancreas of transgenic mice, recapitulating the expression pattern of the endogenous nephrin gene. In this present study, two truly podocyte-specific promoters were identified that drive transgene expression in podocytes without expression in extrarenal tissues in adult or embryonic mice. A 1.25-kb fragment driving a lacZ reporter gene (p1.25N-nlacF) was derived from murine Nphs1 promoter similar to a human NPHS1 promoter fragment previously reported. Transgenic mice were generated and beta-galactosidase (beta-gal) expression was analyzed. Four of twelve founder mice were found to express beta-gal in podocytes (33% penetrance). Expression in brain and pancreas was absent in all animals, suggesting that nephrin expression in these organs might be driven by distinct cis-regulatory elements that can be removed to obtain podocyte-specific expression. A 2.5-kb fragment derived from the human NPHS2 (podocin) gene was designed in a similar fashion to drive lacZ expression in transgenic mice (p2.5P-nlacF). Twelve of twlve NPHS2 mouse founder lines expressed beta-gal exclusively in podocytes (100% penetrance). Beta-gal activity was not observed extrinsic to the kidney in p1.25N-nlacF or p2.5P-nlacF mouse embryos at gestational time points between 8.5 d post coitus and birth. In conclusion, the 2.5-kb NPHS2 promoter fragment may be useful for podocyte-specific transgenic expression when extrarenal expression of a transgene is problematic.

Caloric restriction (CR) without malnutrition is the only experimental manipulation that has consistently been shown to increase the mean and maximum lifespan of laboratory rodents. It has been suggested that CR extends the longevity of rodents and reduces the incidence of age-related pathological lesions by reducing the levels of DNA damage and mutations that accumulate with age within a cells genome. This hypothesis is attractive because the integrity of the genome is essential to a cell/organism and because it is supported by the observations that both cancer and immunological defects, which increase significantly with age and are delayed by CR, are associated with changes in DNA damage. However, all the evidence supporting the premise that the accumulation of DNA damage/mutations plays a role in aging and CR is correlative, i.e., the anti-aging action of CR-fed rodents is correlated with decreased DNA damage and mutation and increased DNA repair capacity. Therefore, additional experiments are required which employ more accurate assays of the DNA repair pathways as well as genetically engineered animal models to establish the role of specific DNA repair pathways and/or enzymes in the anti-aging action of CR. In this paper, we review the proposed mechanisms of DNA damage/repair while providing insight into current research that may assist in "unlocking" the mechanisms behind the life-prolonging effect of CR.

The utility of promoter fragments isolated from the 5'-flanking region of endogenous mammalian genes to drive transgene expression in vivo is often limited by low expression levels. In this study, a bigenic system was established that allows constitutive overexpression of transgenes in a tissue-specific fashion in transgenic mice in a time- and cost-effective fashion. A modified floxed expression vector was constructed [CMVflox-enhanced green fluorescent protein (eGFP)], in which a lacZ cassette (beta-galactosidase) flanked by lox sites was placed between a CMV-promoter and the transgene of interest (eGFP). Before Cre recombination, expression of eGFP was effectively prevented by the interposed floxed lacZ cassette, whereas beta-galactosidase was strongly expressed in transiently transfected cells. Transcription of the gene of interest (eGFP) could be irreversibly activated by cotransfection with Cre recombinase. Mice transgenic for CMVflox-eGFP were generated by pronuclear injection. A rapid assay was developed to identify transgenic founders with active transgene expression by measuring transgene activity (beta-galactosidase) in tail biopsies. Transgene activity in tails correlated with transgene expression in most other tissues tested including podocytes within the kidney. To activate expression of the gene of interest in a tissue-specific fashion, founder mice were mated to the Cre mouse line 2.5P-Cre previously shown to mediate 100% Cre recombination exclusively in podocytes (Moeller MJ, Sanden SK, Soofi A, Wiggins RC, and Holzman LB. Genesis 35: 39-42, 2003). In doubly transgenic offspring, high-level eGFP expression resulting from Cre excision of the interposed lacZ cassette was detected in four of seven CMVflox-eGFP founder lines. This approach should also circumvent common limitations arising from lethality or transgene silencing as a consequence of transgene overexpression.