Ipc

restriction enzyme-cleaved PCR product

HlnD III Pat I

HlnD III Pat I

Fig. 1. Construction of an oxidative stress-inducible reporter transgene. The putative promoter region of the paraquat-inducible gene K08F4.7 was recovered by PCR, using cosmid K08F4 as a template and primers targeted immediately upstream of the K08F4.7 and K08F4.6 putative initiator ATG codons. Incorporated into the 5' portion of these primers were restriction site sequences for Hindlll and Pstl (underlined). After purification, this PCR fragment was cleaved with HindlU and Pstl and the "sticky end" product was recovered by gel purification, and ligated into a similarly purified ifmdIII/i'iil-linearized pPD95.69 vector fragment.

GFP coding sequence

Fig. 1. Construction of an oxidative stress-inducible reporter transgene. The putative promoter region of the paraquat-inducible gene K08F4.7 was recovered by PCR, using cosmid K08F4 as a template and primers targeted immediately upstream of the K08F4.7 and K08F4.6 putative initiator ATG codons. Incorporated into the 5' portion of these primers were restriction site sequences for Hindlll and Pstl (underlined). After purification, this PCR fragment was cleaved with HindlU and Pstl and the "sticky end" product was recovered by gel purification, and ligated into a similarly purified ifmdIII/i'iil-linearized pPD95.69 vector fragment.

designed to amplify the sequence between the K08F4.6 and K08F4.7 ATG initiator codons, and to introduce 5' extensions containing convenient restriction enzyme sites. The promoter region is amplified from cosmid K08F4 by AmpliTaq Gold (PerkinElmer, Norwalk, CT) polymerase, as per the manufacturer protocol. [When possible, we amplify from cosmid clones to minimize amplification cycles and the likelihood of introducing PCR-generated mutations. We also more typically use high-fidelity polymerases such as Pfu (New England BioLabs, Beverly, MA).] The completed reaction is purified by passage through a PCR purification column (Qiagen, Chatsworth, CA), and then the PCR product is digested with ffindlll and Pstl, and gel purified (Qiagen gel extraction kit). As a GFP expression vector, we have chosen pPD95.69 from the Fire collection. This is a promoterless expression vector that contains a GFP-coding region with the S65C substitution and five small introns. This vector is cleaved with Hindlll and Pstl, gel purified, and ligated to the promoter fragment. After transformation, clones are recovered and checked by restriction digest, resulting in the identification of an appropriate clone, designated pAF15.

Recovery of Transgenic Lines pAF15 is introduced into wild-type C. elegans animals by gonad microinjection, initially using the dominant morphological marker rol-6(sul006) encoded in coinjected plasmid pRF4'1 to identify transgenic animals independent of any GFP expression from the pAF15 reporter transgene. Microinjection solutions contain pAF15 (100 ng//xl) and pRF4 (100 ng//xl). [Gonad microinjection leads to the incorporation of injected DNA into germ cells and a fraction of the resulting progeny animals. These transformed Fi progeny can be identified by the abnormal movement "Roller" phenotype they exhibit. A fraction of the transformed Fi animals will contain heritable extrachromosomal arrays containing multiple copies of both injected plasmids, and thus will segregate transgenic Roller F2 progeny, allowing the establishment of heritable (but mitotically and meiotically unstable) transgenic lines.]

After subsequently demonstrating oxidative stress-dependent induction of the pAF15 reporter transgene (see below), additional lines are generated by injecting only pAF15 and directly selecting transgenic animals on the basis of oxidative stress-induced GFP expression, using a Leica (Bensheim, Germany) MZ12 dissecting microscope equipped with epifluorescence optics to screen progeny of injected animals. One transmitting extrachromosomal line recovered in this manner, CL1166, has been used to generate completely stable chromosomally integrated lines. This is accomplished by exposing 20 transgene-bearing young adult animals to 7000 rad of y rays from a cesium-66 source, and then cloning

11 C. C. Mello, J. M. Kramer, D. Stinchcomb, and V. Ambros, EMBOJ. 10, 3959 (1991).

each exposed animal. In our work, 10 transgene-bearing first-generation progeny animals were subsequently cloned from each parental animal. After production of second-generation (F2) animals, 200 plates are exposed to hyperbaric oxygen, and plates are screened for high proportions (>75%) of GFP-expressing F2 animals, indicative of transgene stabilization via chromosomal insertion. F2 animals are cloned from candidate integrated clones, and clones are identified in which all F3 progeny express GFP in response to hyperbaric oxygen exposure. In our work, three completely stable lines were recovered and outcrossed to wild-type animals (six times for strain CL2166). Chromosomal integration of the transgene is confirmed by demonstrating appropriate Mendelian segregation of the transgene.

Hyperbaric Oxygen as Oxidative Stress

Redox quinones such as paraquat and juglone can generate intracellular superoxide, and have been routinely used in C. elegans to generate oxidative stress.4,1213 However, these compounds are both unstable and highly toxic, making them difficult to use to induce nonlethal oxidative stress. We therefore apply hyperbaric oxygen exposure to generate oxidative stress in C. elegans, using a cylindrical pressure vessel (16 x 17.5 cm external dimensions) custom machined from stainless steel (see Fig. 2). We have found that whereas longer exposures (e.g., 48 hr) to 40-psi oxygen (99.95% industrial grade) are lethal to C. elegans, shorter exposures (up to 8 hr) do not reduce the overall life span of wild-type animals, but can induce oxidative stress. We have therefore generally used hyperbaric oxygen to assay reporter responses.

Assaying Oxidative Stress Induction of Reporter Transgenes

Three independent chromosomally integrated transgenic lines carrying pAF15 (CL2166, CL3166, and CL4166) are exposed to a variety of oxidative stresses. All these lines show a low level of GFP expression in most tissues under normal growth conditions. (The basal GFP expression in these lines increases when animals are grown at higher temperatures, possibly reflecting increased endogenous oxidative stress in animals with higher metabolic rates.) This baseline level of GFP expression is strongly increased by a 4-hr exposure to hyperbaric oxygen (see Fig. 3A). To quantify transgene induction, cohorts of treated or untreated young adult animals are individually digitally imaged for GFP expression, using an Axioskop epifluorescence microscope (Zeiss, Thornwood, NY) equipped with a Cohu (San Diego, CA) monochrome camera, and the mean pixel density (corresponding to overall mean GFP expression in each animal) is determined with NIH

12 N. Iishi, K. Takahashi, S. Tomita, T. Keino, S. Honda, K. Yoshino, and K. Suzuki, Mutat. Res. 237, 165 (1990).

Fig. 2. Apparatus for exposure to hyperbaric oxygen. A stainless steel pressure vessel (PV) was connected to the regulator valve (TR) of a standard oxygen tank (OT) containing 99.95% pure oxygen (industrial grade) with vacuum tubing. Animals to be exposed to hyperbaric oxygen were propagated on solid agar medium petri plates in ambient air, and then placed inside the vessel. After sealing the vessel, air was purged with oxygen by opening the inlet (IV) and outlet (OV) valves, and allowing pure oxygen to flow through the vessel for 1 min. The outlet valve was then closed, and the subsequent increasing pressure was monitored with the in-line pressure gauge (PG). When the desired pressure was obtained (typically 40 psi), the tank regulator and intake valves were simultaneously closed. Line pressure was released by opening the line release valve (RV), and the pressure vessel was removed from the line and placed in a 20° incubator. At the conclusion of hyperbaric oxygen exposure, vessel pressure was released by partial opening of the output valve, which resulted in a relatively slow depressurization (i.e., >2 min) of the vessel.

Fig. 2. Apparatus for exposure to hyperbaric oxygen. A stainless steel pressure vessel (PV) was connected to the regulator valve (TR) of a standard oxygen tank (OT) containing 99.95% pure oxygen (industrial grade) with vacuum tubing. Animals to be exposed to hyperbaric oxygen were propagated on solid agar medium petri plates in ambient air, and then placed inside the vessel. After sealing the vessel, air was purged with oxygen by opening the inlet (IV) and outlet (OV) valves, and allowing pure oxygen to flow through the vessel for 1 min. The outlet valve was then closed, and the subsequent increasing pressure was monitored with the in-line pressure gauge (PG). When the desired pressure was obtained (typically 40 psi), the tank regulator and intake valves were simultaneously closed. Line pressure was released by opening the line release valve (RV), and the pressure vessel was removed from the line and placed in a 20° incubator. At the conclusion of hyperbaric oxygen exposure, vessel pressure was released by partial opening of the output valve, which resulted in a relatively slow depressurization (i.e., >2 min) of the vessel.

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