Normal adult male Sprague-Dawley rats (Hilltop Laboratories, Philadelphia, PA) were maintained on a 12L:12D cycle with free access to food and water in the University of Virginia vivarium. All experiments complied with the regulations set forth by the Animal Welfare Act (Public Law 91-579), the Guide for the Care and Use of Laboratory Animals (NRC, 1996) published by the Department of Health and Human Services, and the policies and procedures of the University of Virginia Institutional Animal Care and Use Committee.
Preparation of Plasmids
The cres promoter constructs were derived from the mouse promoter, which is similar in structure to the rat promoter and were previously described. The GGT promoter IV constructs p135LUC, p250LUC, p530LUC, p903LUC, p1976LUC, and p(-16 to -36)135LUC were described previously. The inserts from these vectors were transferred from pGL3-enhancer to pGL3-basic (both from Promega Biosciences Inc., Madison, WI) using an NheI/Narl restriction digest. The resulting plasmids were named pGL3-b135, pGL3-b250, pGL3-b530, pGL3-b903, pGL3-b1976, and pGL3-b135del, respectively. To generate GGT promoter IV-EGFP, pEGFP-C3 (BD Biosciences Clontech, Palo Alto, CA) was restriction digested with NarI and XbaI. The enhanced green fluorescent protein (EGFP) fragment was then ligated into p1976LUC that had been restricted with NarI and XbaI, releasing the luciferase sequence except for 33 bp encoding the first 11 amino acids. To generate pGL3-b681, pGL3-903 was digested with MsH and Xmnl to produce a 716-bp fragment that was cloned into pGL3-basic digested with Smal.
To generate pGL3-b6500, a X phage clone-containing genomic sequence between GGT exon V and gGt promoter IV, kindly provided by Dr. Y. Laperche (INSERM, Creteil, France), was digested with XbaI and BgHl. A 5-kb fragment was ligated into pGL3-1976 digested with NheI and BgKI. The 5-kb X phage fragment overlapped with approximately 500 bp at the 5′ end of pGL3-b1976; thus, pGL3-b6500 contained an additional 4.5 kb of sequence 5′ to the 2 kb previously published under Genbank accession number AF218050. To generate pGL3-b135mut, pGL3-b530m1-3, andpGL3-b530c, approximately 50 ng of pGL3-b135 and pGL3-b530 were subjected to PCR-based mutagenesis using the QuikChange mutagenesis kit (Stra-tagene, La Jolla, CA). The PCR reactions (14 cycles of 950C for 30 sec, 550C for 1 min, and 680C for 11 min) were done using the following pairs of primers. Bold represents the nucleotides that were mutated with the original nucleotides above the sequence.
All plasmids were confirmed by sequencing (Biomolecular Research Facility, University of Virginia, Charlottesville, VA). All plasmids used for in vivo electroporation were purified by EndoFree Plasmid Maxi Kit (Qiagen, Valencia, CA) or CsCl density gradient ultracentrifugation.
In Vivo Electroporation
Rats were anesthetized with an intraperitoneal pentobarbital sodium injection (Nembutal; Abbott Laboratories, Chicago, IL). The scrotal contents were reached by a midline abdominal incision. Micropipettes made from R-6 glass with an outer diameter of 0.9 mm and an inner diameter of 0.6 mm (Drummond Scientific Company, Broomall, PA) were pulled on a Flaming/Brown micropipette puller (Model P-97; Sutter Instrument Company, Novato, CA). The pipettes were then sharpened on soapstone to a tip diameter of 20-30 |xm. For intraluminal injections, 2-5 |xl of either pEGFP-N1 (BD Biosciences Clontech) or GGT promoter IV-EGFP at 2 |xg/^l were injected into the lumen of an initial segment tubule. For GGT promoter IV analysis, 15 |xl of DNA were injected into zones 1a and 1b (see for characterization of zones) of the initial segment interstitium underneath the capsule. For cres promoter analysis, 25 ^l of DNA were injected into zones 1a, 1b, and 1c of the initial segment. Following the injection, the tissue was grasped between the plates (7-mm diameter) of a pair of tweezertrodes (BTX, San Diego, CA), and 8 X 50-msec pulses of 21-24 V were delivered to the tissue using an Electro Square Porator ECM 830 (BTX). The distance between the electrodes was kept constant at 0.2 cm. The testis and epididymis were returned to the scrotum, and the body wall and skin were sutured. At the indicated times, the rats were euthanized with carbon dioxide gas, and the tissue was removed, frozen in liquid nitrogen, and stored at -800C until use. The entire initial segment was taken from rats injected with cres promoter constructs, whereas only the proximal half of the initial segment was taken from rats injected with GGT promoter IV constructs.
To examine the different length GGT promoter IV constructs, equi-molar amounts of DNA were used as follows: pGL3-b135 at 2.5 |xg/^l, pGL3-b250 at 2.6 |g/|l, pGL3-b530 at 2.7 |g/|l, pGL3-b681 at 2.8 |g/ |Д pGL3-b903 at 2.9 |g/|l, and pGL3-b1976 at 3.4 |g/|l. For the experiments comparing pGL3-b530 and pGL3-b6500, 2.5 |g/|l of pGL3-b6500 and 1.2 |g/|l of pGL3-b530 were used. Linearized pUC18 was added to make the total DNA mass equivalent for each injection within an experiment. To control for electroporation efficiency, 1 ng/|l of pRL-SV40 was included with every injection. To examine the cres promoter constructs, 3 |g/|l of the different cres constructs in pGL3-basic were coinjected with 50 ng/|l of pRL-TK (electroporation efficiency control).
Following electroporation, tissues were removed at 72 h and frozen in liquid nitrogen. Five-micron frozen sections were prepared by the University of Virginia Cell Science Core, and sections were analyzed with a Zeiss 410 Laser Scanning Confocal Microscope (Carl Zeiss Optical, Inc., Thornwood, NY). Integrated black-and-white images were captured and then pseudocolored.
Dual Luciferase Assay
Assays were performed with the Dual Luciferase Assay kit from Pro-mega according to the manufacturer’s instructions with minor modifications. Previously collected and frozen tissues were ground with a mortar and pestle under liquid nitrogen. Once the liquid nitrogen had evaporated, the pellets were resuspended in Luciferase Assay Reagent II. Protein determinations were made using the Bradford method (Bio-Rad Laboratories, Hercules, CA), and 50 |g of protein in 20 |l were assayed. Measurements were taken on an FB-15 luminometer (Zylux Corporation, Maryville, TN). For GGT experiments, background values were determined on the contralateral initial segments that were uninjected and unelectroporated. Background values were then subtracted from each sample, and the ratio of firefly to renilla luciferase was calculated by dividing the firefly value of each sample with the respective renilla value. For cres experiments, background values from age-matched controls were subtracted, and then ratios were calculated as described for GGT.
Tags: epididymis, gene regulation, male reproductive tract