Abstract
Introduction:
Fibrillin-1 deficient mice (mgR/mgR) are accepted as model for Marfan syndrome. However, methods to safely detect homozygous mgR/mgR remain uncertain and complicated. In this study we present safe genotyping of mgR/mgR by Realtime quantative-PCR (qRT-PCR) ascertained by phenotype characterization.
Methods:
Heterozygous female (mgR/+) were matched with homozygous male (mgR/mgR). DNA was isolated from tags with standard-methods. QRT-PCR was performed with 4 primer [Alb1-e12_MUS, qNEO1, qNEO2, qNEO3] detecting the native fibrillin-1 gene or the introduced neomycin cassette, respectively. Data was interpreted with 2(delta-delta-CT)-method. Standard dCT and RQ-limit for homozygous mice were defined mathematically. Out of 383 littermates of heterozygous-homozygous-matings primer were compared. Necropsy was performed on 50 male mice that naturally died to define phenotypic correlation.
Results:
Primer neo2 turned out to be the most reliable and valuable primer to define genotype. Standard dCT(neo2) was 0.46. RQ-values of mgR/mgR had normal cause of distribution (RQ*100: mean:101.56, SD:21.30). Therefore threshold was set RQ (neo2)=0.59. With this method, 2.5% of homozygous animals show RQ (neo2)<0.59 and are falsely defined as heterozygous. During necropsy 50/50 mice turned out to be phenotypically marfanoid. 49/50 died due to spontaneous aortic rupture. 1/50 died for unknown reason with diaphragmatic hernia and impingement by intestines. 49/50 previously had been analyzed as homozygous mgRmgR, 1/50 as heterozygous showing RQ(neo2)=0.50.
Conclusions:
Standardized qRT-PCR using internal standard and threshold as presented results in safe genotype-phenotype matching. A certain and easy method to safely genotype homozygous mgR/mgR is pivotal to ensure high-value investigation of this promising model of Marfan syndrome.