Expression of cytochrome P 450 1 b 1 ( Cyp 1 b 1 ) during early murine development

Cytochrome P4501B1 ( CYP1B1) gene [1,2] encodes a monooxygenase capable of metabolizing both xenobiotics [3] and endogenous compounds such as estrogen, testosterone and retinoids [4-7]. In humans, mutations affecting CYP1B1 are associated with abnormal development of the anterior chamber angle of the eye manifested as aggressive, early onset glaucoma, which most frequently is classified as primary congenital glaucoma [8-11]. Cyp1b1 mice have milder phenotype due to the presence of both normal and abnormal filtration structures [12-14]. In situ hybridization analysis of tissue sections detected Cyp1b1 mRNA in the developing and adult mouse eye at embryonic day 15 (E15) and postnatal days 4, 7, and 30 [15]. PCR based assay of whole mouse embryo cDNA suggested that Cyp1b1 is expressed as early as E11 [16]. The objective of this study was to examine Cyp1b1 expression domains during the early stages of murine development by whole mount in situ hybridization. METHODS Animals: Male and female FVB/NCrlBR mice were purchased from Charles River Laboratories (Wilmington, MA). For timed mating experiments breeding units (one male and two females) were set at 3.30-4 PM. Inspection for vaginal plugs was performed before 9 AM on the following day. For staging purposes, noon of the day of vaginal plug was considered 0.5 days post-conception (dpc). On the desired day, animals were euthanized at noon by carbon dioxide inhalation. Embryos from two pregnant females were collected at 9.5 dpc. Embryos from one pregnant female were collected at 10.5 dpc and 11.5 dpc, respectively. The embryos were dissected from the uterus, fixed overnight with 4% paraformaldehyde in phosphate buffered saline (PBS) at 4 °C, dehydrated through a methanol series (25%, 50%, 75%, 100%, and 100%) and stored in 100% methanol at -20 °C. All animal manipulations were conducted in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and were approved by The Animal Care Committee (ACC) of the University of Connecticut Health Center. In situ hybridization: A 848 bp Cyp1b1 cDNA fragment was amplified by polymerase chain reaction (PCR) by using primer set of 5'-AGC TGA GCT CGC TGT CTA CC-3' and ©2004 Molecular Vision