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Mark Christian Butler, Jack M Sullivan; Identifying the Integration Site of the hRhoPro347Ser Gene in the C1 Transgenic Mouse adRP Model. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):240.
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© ARVO (1962-2015); The Authors (2016-present)
Identify and characterize the integration site of the human rhodopsinPro347Ser (hRhoP347S) transgene in the C1 transgenic adRP mouse (Li et al., 1996, PNAS, 93: 14176-81), which is a model for preclinical gene therapeutics testing. This study was prompted by observation of syndactyly in homozygous but not heterozygous C1 transgenic mice.
Genomic DNA was isolated from blood of C1 mice using established protocol. Mapping the C1 integration site was conducted with the Genome Walker Universal Kit (Clontech). PCR amplification occurred with Advantage 2 Polymerase mix or AmpliTaq Gold 2X PCR master mix. Primer 3.1 software was used to design all PCR and DNA sequencing primers. Data analysis was conducted with NCBI standard nucleotide Blast program. DNA electrophoresis was conducted in 1% (w/v) agarose gels in 1X TBE buffer with EtBr staining.
Following examination of C1 and A1 hRhoP347S transgenic adRP animals (C1 has 0.25 ratio of hRho mRNA relative to mouse RHO mRNA and A1 has 1.0 ratio) it was evident that only mice homozygous for C1 exhibited the syndactyly phenotype. Syndactyly was an effect of the integration event and not due to the exogenous DNA since both C1 and A1 lines were created with the same fragment of hRhoP347S genomic DNA. To identify the location of the C1 transgene integration site genome walking was performed. Genome walking experiments returned sequencing results that were used in NCBI BLAST searches that showed hRhoP347S insertion within the mouse Fibrillin-2 gene (FBN2). These results were used to design PCR primers flanking the integration site, which were used to confirm that hRhoP347S integration in the C1 line occurred in the FBN2 gene at intron-7 (nucleotide 138,219). A recent confirmatory publication reported syndactyly in a FBN2 knock-out mouse model (Shi et al., 2013, IOVS, 54: 7163-73).
Identification of the integration site in a transgenic mouse model should be a prerequisite to their use in preclinical therapeutic investigations. Site identification allows phenotypic characterization to be correctly attributed to either the insertion of novel genetic material (e.g. mutant Rho), or mutations of endogenous genes due to random transgene insertion. In addition, knowledge of the insertion site can facilitate phenotypic identification (e.g. syndactyly) of desired transgenic animals for confirmation by genotyping, thus conserving resources.
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