Minigene Assay

Twenty-nine variants close to intron–exon boundary in COL1A1 and COL1A2 from 34 probands were selected for the minigene splicing assay ( Figure 1A ). The fragments of interests varying from 808 bp to 2,510 bp ( Table S1 ) which contain the putative splicing variant along with flanking exons were amplified by high fidelity PCR. The PCR was carried out using HS DNA polymerase (TaKaRa, Shiga, Japan) and forward and reverse primers with restriction sites for BamHI or MluI (New England Biolabs, Ipswich, MA, USA). Primers were designed for each target fragment using Primer3 (http://primer3.ut.ee/) ( Table S1 ). The amplified target fragments were cloned into the pCAS2 vector ( Figure 1B ) using restriction endonucleases BamHI, MluI, and T4 DNA ligase (New England Biolabs). The constructed vector was further transformed into E. coli DH5α Competent Cells (TaKaRa, Shiga, Japan), followed by sequencing verification. Both the purified constructs of wild type and mutant type were transferred into HEK293T cells using Invitrogen Lipofectamine 3000 Transfection Kit (Thermo Fisher Scientific). HEK293T cell line was selected to eliminate endogenous interference for its low expression of type I collagen. After 24 h incubation, RNA was isolated using Trizol reagent (Invitrogen). One microgram total RNA was used for RT-PCR using PrimeScript RT reagent kit with gDNA Eraser (TaKaRa). PCR products were separated on 1% agarose gel containing ethidium bromide. The target DNA bands were purified using GeneJET Gel Extraction Kit (Thermoscientific, Lithuania), followed by DNA sequencing with ABI3730xl (Thermo Fisher Scientific, Waltham, MA, USA). The procedure was summarized in the schematic map ( Figure 1C ).

Schematic map of the procedure of minigene assay. (A) The distribution of mutations identified in COL1A1 and COL1A2 in OI patients. The variants at exon–intron boundary were labeled on the map, including both the annotated variants (shown at the top in black) which can be found in HGMD, as well as novel variants (shown at the bottom in red). The numbers in the cartoon represent the exons within COL1A1 or COL1A2. (B) Overview of pCAS2 (Upper panel) vector constructs. (C) Experimental procedure of minigene assay.