The bacteriophage P1 Cre͞loxP system has become a powerful tool for in vivo manipulation of the genomes of transgenic mice. Although in vitro studies have shown that Cre can catalyze recombination between cryptic ''pseudo-loxP'' sites in mammalian genomes, to date there have been no reports of loxP-site infidelity in transgenic animals. We produced lines of transgenic mice that use the mouse Protamine 1 (Prm1) gene promoter to express Cre recombinase in postmeiotic spermatids. All male founders and all Cre-bearing male descendents of female founders were sterile; females were unaffected. Sperm counts, sperm motility, and sperm morphology were normal, as was the mating behavior of the transgenic males and the production of two-celled embryos after mating. Mice that expressed similar levels of a derivative transgene that carries an inactive Cre exhibited normal male fertility. Analyses of embryos from matings between sterile Cre-expressing males and wild-type females indicated that Cre-catalyzed chromosome rearrangements in the spermatids that lead to abortive pregnancies with 100% penetrance. Similar Cre-mediated, but loxP-independent, genomic alterations may also occur in somatic tissues that express Cre, but, because of the greater difficulty of assessing deleterious effects of somatic mutations, these may go undetected. This study indicates that, following the use of the Cre͞loxP site-specific recombination systems in vivo, it is prudent to eliminate or inactivate the Cre recombinase gene as rapidly as possible.T he bacteriophage P1 recombinase, Cre, is a member of the integrase family of site-specific recombinases that catalyzes recombination between loxP DNA elements as a part of the normal viral life cycle (1, 2). All members of the integrase family share a similar mechanism of action (3). In Cre-mediated recombination, individual loxP sites are bound by Cre homodimers (4). Two Cre-bound loxP sites interact in the recombination reaction. The loxP site is a 34-bp element consisting of two 13-bp inverted repeats separated by a directional 8-bp core (1, 5-7). During cleavage, Cre Tyr 324 becomes covalently linked to the 3Ј-phosphate, forming a DNA-phosphotyrosine intermediate (8, 9). Interaction with a second loxP-bound Cre dimer allows nucleophilic attack of the 3Ј-phosphotyrosine in one loxP site by the free 5Ј-hydroxyl of the other. This ligation reaction induces a structural rearrangement of the tetrameric complex, allowing the same reaction to occur on the second strand of each loxP site, thus completing the strand exchange reaction (8,(10)(11)(12).Cre has proven to be a valuable tool for manipulating the genomes of mammalian cells and of mice in situ (13-19). As an example, Cre-mediated recombination has all but eliminated the problem of ''selection cassette effects'' associated with some targeted mutations (15). Although Cre is most commonly used to generate short deletions in transgenic mice (13), it has also proven useful for recombination between sites up to several centimorgans apart on individual c...
In vitro studies suggest that collecting duct-derived (CD-derived) endothelin-1 (ET-1) can regulate renal Na reabsorption; however, the physiologic role of CD-derived ET-1 is unknown. Consequently, the physiologic effect of selective disruption of the ET-1 gene in the CD of mice was determined. Mice heterozygous for aquaporin2 promoter Cre recombinase and homozygous for loxP-flanked exon 2 of the ET-1 gene (called CD-specific KO of ET-1 [CD ET-1 KO] mice) were generated. These animals had no CD ET-1 mRNA and had reduced urinary ET-1 excretion. CD ET-1 KO mice on a normal Na diet were hypertensive, while body weight, Na excretion, urinary aldosterone excretion, and plasma renin activity were unchanged. CD ET-1 KO mice on a high-Na diet had worsened hypertension, reduced urinary Na excretion, and excessive weight gain, but showed no differences between aldosterone excretion and plasma renin activity. Amiloride or furosemide reduced BP in CD ET-1 KO mice on a normal or high-Na diet and prevented excessive Na retention in salt-loaded CD ET-1 KO mice. These studies indicate that CD-derived ET-1 is an important physiologic regulator of renal Na excretion and systemic BP. IntroductionEndothelin-1 (ET-1) was initially described as a potent endothelial cell-derived vasoconstrictor (1); however, the peptide is now known to be produced by many cell types and to elicit multiple biologic effects (2). The kidney is likely an important target; ET-1 causes renal vasoconstriction, mesangial cell contraction, glomerular cell proliferation, ECM accumulation, and alterations in nephron fluid and electrolyte transport (2). While many renal cell types synthesize and bind ET-1, the collecting duct (CD) is of particular importance: The renal inner medulla contains the highest concentration of ET-1 in the body (3), and the inner medullary CD (IMCD) is the predominant renal site of ET-1 production (4-8) and receptor expression (9-11).In vitro studies suggest that ET-1 inhibits Na and water reabsorption in the cortical CD (CCD) and IMCD and that this occurs through activation of the ET B receptor (ETRB). ET-1 inhibits vasopressin-stimulated (AVP-stimulated) water flux in isolated CCD (9, 12) and reduces AVP-stimulated cyclic AMP accumulation (13-15) and osmotic water permeability (16, 17) in isolated IMCDs. ET-1 also inhibits mineralocorticoid and AVP-stimulated Na and Cl reabsorption in isolated CCDs (12,18,19) and decreases Na/K-ATPase activity in suspensions of IMCDs (20). Despite these data, demonstrating such an ET-1 effect in vivo and clarifying how CD-derived ET-1 physiologically regulates Na and water transport has been problematic. This difficulty stems, in part, from
Aims: To estimate the risk of aphakic glaucoma after lensectomy for congenital cataract and its association with surgery within the first month of life. Method: A retrospective case notes review was conducted of all patients who had lensectomy for congenital cataract during their first year of life at Great Ormond Street Hospital between 1994 and 1997. Patients with pre-existing glaucoma, anterior segment dysgenesis, and Lowe syndrome were excluded. The risk of aphakic glaucoma after surgery was estimated using Kaplan-Meier survival analysis. Results: 80 patients, undergoing 128 lensectomies were eligible. Of these, six patients (nine eyes) were lost to follow up. Based on eye count, the risk of glaucoma by 5 years after lensectomy was 15.6% (95% CI 10.2 to 23.4). Based on patient count, the 5 year risk of glaucoma in at least one eye following bilateral surgery was 25.1% (95% CI 15.1 to 40.0). The incidence of glaucoma remained at a constant level for the first 5 years after surgery. After early bilateral lensectomy, within the first month of life, the 5 year risk of glaucoma in at least one eye was 50% (95% CI 27.8 to 77.1) compared to 14.9% (95% CI 6.5 to 32.1) with surgery performed later (log rank test, p = 0.012). There was no significant difference (KolmogorovSmirnov test: unilateral lensectomy p = 0.587, bilateral lensectomy p = 0.369) in 5 year visual outcomes between eyes operated before and after 1 month of age. Conclusion: Bilateral lensectomy during the first month of life is associated with a higher risk of subsequent glaucoma than with surgery performed later. The reason for this is unclear but it may be prudent, in bilateral cases, to consider delaying surgery until the infant is 4 weeks old. As the incidence of glaucoma is similar for each year after surgery, long term glaucoma surveillance is mandatory.A phakic glaucoma is an established complication following lensectomy for congenital cataract.1-5 It presents as early angle closure glaucoma caused by vitreous pupillary block, inflammatory peripheral anterior synechiae, 6-8 or proliferating retained lens matter. More commonly, it presents as open angle glaucoma up to years after surgery.3 4 6-11 The risk of aphakic glaucoma may be higher with infants operated during the first year of life. 6 12 13 As glaucoma in children can be difficult to manage, parents should be counselled about its likelihood and consequences. The reported prevalence of postoperative glaucoma varies between 6% and 26% of eyes (15% to 45% of patients) among children operated before, as well as after, 1 year of age. 12 14-19 With widespread screening of neonates for congenital cataract, affected infants are increasingly referred by 3 months of age. 20 Thus, it is important to reexamine the risk of postoperative glaucoma specifically within this subgroup of infants.The timing of congenital cataract surgery can have a major impact on long term prognosis. Surgery after 6 weeks of age may deprive the neonatal visual system of vital stimuli during the critical period.21 Surger...
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