Y Mountain Laboratory (RML) prions. No differences were observed in the

Y Mountain Laboratory (RML) prions. No differences were observed in the onset, progression, or histopathology of disease. This indicates that altered MGRN1 function has little or no role in the pathogenesis of transmissible prion diseases and Licochalcone A indirectly supports a role for plasma membrane PrPSc.Mgrn1 expressionMgrn1 expression in the brains of Tg+ and Tg2 mice that were homozygous wild-type at the Mgrn1 locus or heterozygous for the null allele (Mgrn1md2nc/+) was determined by quantitative RTPCR. Tg+ mice were obligate heterozygotes for the Tg(Mgrn1I)C3Tmg transgene since they were generated by mating Tg+; Mgrn1md2nc/+ animals to Tg2; Mgrn1+/+ mice. Brain RNA was extracted from 3 mice of each genotype using TriPure reagent (Roche), followed by DNaseI treatment (Invitrogen) and cDNA synthesis (High Capacity cDNA Kit, Applied Biosystems, Foster City, CA) prior to amplification using SYBR green Brilliant II PCR master mix (Agilent) on a BioRad Opticon II. All samples were analyzed in triplicate on a single plate. Expression was normalized against glucose phosphate isomerase (Gpi) levels using the comparative Ct method to determine the relative quantification value [14]. Mgrn1 primers: GATCTACGGCATCGAGAACAA and AGTGTGTCCCGCAGGTC. Gpi primers: CAACTGCTACGGCTGTGAGA and CTTTCCGTTGGACTCCATGT.Materials and Methods Ethics statementAll animal procedures adhered to Association for Assessment and Accreditation of Laboratory Animal Care guidelines and were approved by the Institutional Animal Care and Use Committee of the McLaughlin Research Institute.Prion inoculationsAll procedures involving animals adhered to Association for Assessment and Accreditation of Laboratory Animal Care guidelines and were reviewed and approved by the Institutional Animal Care and Use Committee of the McLaughlin Research Institute. Female mice of the following genotypes were inoculated intracerebrally at 36?0 days of age with RML SIS 3 prions using 10 ml of 10 brain homogenate from clinically ill mice: Tg2; Mgrn1md2nc/+ (n = 4), Tg2; Mgrn1+/+ (n = 7), Tg+; Mgrn1md2nc/+Figure 1. Kaplan-Meier plots for health status following RML prion inoculation. (A) Plot indicating proportion of healthy Mgrn1md2nc/+ and Mgrn1md2nc/md2nc animals over time following RML prion inoculation. (B) Plot showing survival of Mgrn1 transgenic (Tg+) and non-transgenic (Tg2) Mgrn1md2nc/+ and Mgrn1+/+ mice over time following inoculation with RML prions. doi:10.1371/journal.pone.0055575.gMGRN1 Levels Do Not Influence Prion DiseaseTable 2. Disease progression.Genotype (n) Tg2; Mgrn1md2nc/+ (4) Tg2; Mgrn1+/+ (7) Tg+; Mgrn1md2nc/+ (6) Tg+; Mgrn1+/+ (8)Average age at inoculation (range) 53 (40?9) 47 (39?0) 41 (36?0) 45 (37?9)Average number of days post-inoculation to appearance of paresis (range) (SD)a 103 (98?07) (6.4) 103 (88?07) (11.6) 110 (94?15) (11.3) 102 (94?11) (7.4)Average number of days post-inoculation to death or euthanasia (range) (SD)a 148 (141?52) (5.3) 138 (127?60) (11.3) 142 (135?46) (5.6) 141 (120?52) (9.5)a No statistically significant differences by one-way ANOVA (all p.0.40). doi:10.1371/journal.pone.0055575.t(n = 6) and Tg+; Mgrn1+/+ (n = 8). An independent cohort of 4 female and 3 male Mgrn1md2nc/+ and 1 female and 8 male Mgrn1md2nc/md2nc mutant mice were also inoculated with RML prions; in these studies, Mgrn1md2nc/+ mice were used as controls since they are phenotypically normal and a coisogenic Mgrn1+/+ line was not available at the time. Animals were monitored daily for general healt.Y Mountain Laboratory (RML) prions. No differences were observed in the onset, progression, or histopathology of disease. This indicates that altered MGRN1 function has little or no role in the pathogenesis of transmissible prion diseases and indirectly supports a role for plasma membrane PrPSc.Mgrn1 expressionMgrn1 expression in the brains of Tg+ and Tg2 mice that were homozygous wild-type at the Mgrn1 locus or heterozygous for the null allele (Mgrn1md2nc/+) was determined by quantitative RTPCR. Tg+ mice were obligate heterozygotes for the Tg(Mgrn1I)C3Tmg transgene since they were generated by mating Tg+; Mgrn1md2nc/+ animals to Tg2; Mgrn1+/+ mice. Brain RNA was extracted from 3 mice of each genotype using TriPure reagent (Roche), followed by DNaseI treatment (Invitrogen) and cDNA synthesis (High Capacity cDNA Kit, Applied Biosystems, Foster City, CA) prior to amplification using SYBR green Brilliant II PCR master mix (Agilent) on a BioRad Opticon II. All samples were analyzed in triplicate on a single plate. Expression was normalized against glucose phosphate isomerase (Gpi) levels using the comparative Ct method to determine the relative quantification value [14]. Mgrn1 primers: GATCTACGGCATCGAGAACAA and AGTGTGTCCCGCAGGTC. Gpi primers: CAACTGCTACGGCTGTGAGA and CTTTCCGTTGGACTCCATGT.Materials and Methods Ethics statementAll animal procedures adhered to Association for Assessment and Accreditation of Laboratory Animal Care guidelines and were approved by the Institutional Animal Care and Use Committee of the McLaughlin Research Institute.Prion inoculationsAll procedures involving animals adhered to Association for Assessment and Accreditation of Laboratory Animal Care guidelines and were reviewed and approved by the Institutional Animal Care and Use Committee of the McLaughlin Research Institute. Female mice of the following genotypes were inoculated intracerebrally at 36?0 days of age with RML prions using 10 ml of 10 brain homogenate from clinically ill mice: Tg2; Mgrn1md2nc/+ (n = 4), Tg2; Mgrn1+/+ (n = 7), Tg+; Mgrn1md2nc/+Figure 1. Kaplan-Meier plots for health status following RML prion inoculation. (A) Plot indicating proportion of healthy Mgrn1md2nc/+ and Mgrn1md2nc/md2nc animals over time following RML prion inoculation. (B) Plot showing survival of Mgrn1 transgenic (Tg+) and non-transgenic (Tg2) Mgrn1md2nc/+ and Mgrn1+/+ mice over time following inoculation with RML prions. doi:10.1371/journal.pone.0055575.gMGRN1 Levels Do Not Influence Prion DiseaseTable 2. Disease progression.Genotype (n) Tg2; Mgrn1md2nc/+ (4) Tg2; Mgrn1+/+ (7) Tg+; Mgrn1md2nc/+ (6) Tg+; Mgrn1+/+ (8)Average age at inoculation (range) 53 (40?9) 47 (39?0) 41 (36?0) 45 (37?9)Average number of days post-inoculation to appearance of paresis (range) (SD)a 103 (98?07) (6.4) 103 (88?07) (11.6) 110 (94?15) (11.3) 102 (94?11) (7.4)Average number of days post-inoculation to death or euthanasia (range) (SD)a 148 (141?52) (5.3) 138 (127?60) (11.3) 142 (135?46) (5.6) 141 (120?52) (9.5)a No statistically significant differences by one-way ANOVA (all p.0.40). doi:10.1371/journal.pone.0055575.t(n = 6) and Tg+; Mgrn1+/+ (n = 8). An independent cohort of 4 female and 3 male Mgrn1md2nc/+ and 1 female and 8 male Mgrn1md2nc/md2nc mutant mice were also inoculated with RML prions; in these studies, Mgrn1md2nc/+ mice were used as controls since they are phenotypically normal and a coisogenic Mgrn1+/+ line was not available at the time. Animals were monitored daily for general healt.