In the injected DNA as an extra-chromosomal multiply array of variable mitotic and meiotic stability. The transmitting lines established for this study have 70?0 meiotic stability. Thus transgenic worms produce both transgenic and non-transgenic progeny. Worms were maintained and propagated at 20uC on solid Nematode Growth Medium (NGM) seeded with OP50 E. coli (Caenorhabditis Genetics Center, University of Minnesota, USA) as food. To prepare age-synchronized animals, nematodes were transferred to fresh NGM plates after reaching maturity at 3 days of age and allowed to lay eggs overnight. Isolated hatchlings from the synchronized eggs (day 1) were cultured on fresh NMG plates at 20uC.Genotype characterizationRNA from adult transgenic worms was prepared using the RNeasy Mini kit (QIAzol Lysis Reagent, Qiagen) and quantified using the NanoDrop apparatus (ThermoScientific). Total RNA was reverse transcribed into cDNA with random primers (Random Hexamers, Applied Biosystems) and the GeneAmp RNA PCR Core kit (Applied Biosystems). A quantitative SPDB cost real-time PCR was performed with Mx3000P QPCR System (Stratagene) using the SYBR Green gene expression assay (Applied Biosystems, AB). Relative quantification of mRNA level was determined using two endogenous SR3029 manufacturer standard gene controls, i.e. peripheral myelin gene PMP-22/GAS-3 and cell division cycle 42 (cdc-42, GTPC. elegans Models for b2-m Amyloidosisbinding protein) according to Hoogewijs [23] and, data analysis was performed with MxPro QPCR Software (Stratagene). All measurements were determined in triplicate. Data points collected correspond to the number of PCR cycles (Ct value) required for the fluorescent signal to cross the detection threshold of the thermal cycler. Ct values were normalized to correct for minor differences in cDNA concentrations by subtracting the average of the Ct values of the reactions in triplicate of each transgenic strain from the geometric mean of Ct values of cdc-42 reactions, and analyzed using the comparative 22DDCt method [24].Larval growthOne hundred synchronized eggs were plated on fresh NMG plates seeded with OP50, left at 20uC and the number of worms at L1/L2, L2/L3 and L4/adult larval stage were scored after 13, 22 and 40 hours, respectively.Life-spanGravid worms were allowed to lay eggs for 3? hours at 20uC, to produce an age-synchronized population. Once the worms reached their reproductive maturity, they were transferred daily until the cessation of egg lying to avoid overlapping generations. Nematodes were transferred every day and their viability monitored until all the worms were scored as dead when they failed to display touch-provoked movement.b2-m expressionTransgenic worms were collected with M9 buffer, transferred to tubes, centrifuged and washed twice to remove bacteria. The pellet containing worms was resuspended in lysis buffer (25 mM Tris, pH 7.5, containing 5 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, DTT, and protease inhibitor mixture) and homogenized by sonication. For dot-blot analysis, equal amounts of proteins from homogenized samples (5 mg) were spotted onto nitrocellulose membranes (Millipore), blocked with 5 mM phosphate buffered solution, pH7.4 containing 0.1 Tween 20 (PBS-T) and incubated overnight with a rabbit polyclonal anti-human b2-m antibody (1:1000 dilution, Dako) or 18325633 a rabbit polyclonal antibody recognizing high molecular weight oligomers (A11, 1:1000 dilution, Biosource, USA). Anti-rabbit IgG peroxidase conjugate (1:10000 dilutio.In the injected DNA as an extra-chromosomal multiply array of variable mitotic and meiotic stability. The transmitting lines established for this study have 70?0 meiotic stability. Thus transgenic worms produce both transgenic and non-transgenic progeny. Worms were maintained and propagated at 20uC on solid Nematode Growth Medium (NGM) seeded with OP50 E. coli (Caenorhabditis Genetics Center, University of Minnesota, USA) as food. To prepare age-synchronized animals, nematodes were transferred to fresh NGM plates after reaching maturity at 3 days of age and allowed to lay eggs overnight. Isolated hatchlings from the synchronized eggs (day 1) were cultured on fresh NMG plates at 20uC.Genotype characterizationRNA from adult transgenic worms was prepared using the RNeasy Mini kit (QIAzol Lysis Reagent, Qiagen) and quantified using the NanoDrop apparatus (ThermoScientific). Total RNA was reverse transcribed into cDNA with random primers (Random Hexamers, Applied Biosystems) and the GeneAmp RNA PCR Core kit (Applied Biosystems). A quantitative real-time PCR was performed with Mx3000P QPCR System (Stratagene) using the SYBR Green gene expression assay (Applied Biosystems, AB). Relative quantification of mRNA level was determined using two endogenous standard gene controls, i.e. peripheral myelin gene PMP-22/GAS-3 and cell division cycle 42 (cdc-42, GTPC. elegans Models for b2-m Amyloidosisbinding protein) according to Hoogewijs [23] and, data analysis was performed with MxPro QPCR Software (Stratagene). All measurements were determined in triplicate. Data points collected correspond to the number of PCR cycles (Ct value) required for the fluorescent signal to cross the detection threshold of the thermal cycler. Ct values were normalized to correct for minor differences in cDNA concentrations by subtracting the average of the Ct values of the reactions in triplicate of each transgenic strain from the geometric mean of Ct values of cdc-42 reactions, and analyzed using the comparative 22DDCt method [24].Larval growthOne hundred synchronized eggs were plated on fresh NMG plates seeded with OP50, left at 20uC and the number of worms at L1/L2, L2/L3 and L4/adult larval stage were scored after 13, 22 and 40 hours, respectively.Life-spanGravid worms were allowed to lay eggs for 3? hours at 20uC, to produce an age-synchronized population. Once the worms reached their reproductive maturity, they were transferred daily until the cessation of egg lying to avoid overlapping generations. Nematodes were transferred every day and their viability monitored until all the worms were scored as dead when they failed to display touch-provoked movement.b2-m expressionTransgenic worms were collected with M9 buffer, transferred to tubes, centrifuged and washed twice to remove bacteria. The pellet containing worms was resuspended in lysis buffer (25 mM Tris, pH 7.5, containing 5 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, DTT, and protease inhibitor mixture) and homogenized by sonication. For dot-blot analysis, equal amounts of proteins from homogenized samples (5 mg) were spotted onto nitrocellulose membranes (Millipore), blocked with 5 mM phosphate buffered solution, pH7.4 containing 0.1 Tween 20 (PBS-T) and incubated overnight with a rabbit polyclonal anti-human b2-m antibody (1:1000 dilution, Dako) or 18325633 a rabbit polyclonal antibody recognizing high molecular weight oligomers (A11, 1:1000 dilution, Biosource, USA). Anti-rabbit IgG peroxidase conjugate (1:10000 dilutio.
