Splicing mini-gene reporter assay

The 804-bp genomic fragments spanning 369 bp of intron 1, 249 bp of exon 2, and 186 bp of intron 2, which harbored the two SNPs, were amplified using the specific primer E2 (Additional file: Table S1). The mini-gene reporter assays were constructed by cloning the 804-bp fragments with the wild type (g.19711 T > C and g.19731 G > C; wild haplotype: T-G) or the mutant type (g.19711 T > C and g.19731 G > C; mutant haplotype: C-C) into the empty pSPL3 vector after digestion with EcoRI and XhoI. The constructed vectors and the empty vector were transformed into Trans5α. The clones were cultured on agar containing 100 mg/mL ampicillin and incubated overnight at 37 °C. Positive clones were chosen and incubated overnight at 37 °C in 10 mL of lysogeny broth medium containing 100 mg/mL ampicillin. Plasmids were isolated with the EZNA Endo-Free Plasmid DNA Midi Kit (OMEGA, Cat. #D6915) and then measured by a NanoPhotometer (P330; IMPLEN). The plasmids were sequenced after isolation to guarantee the authenticity and orientation of the vectors.

Bovine mammary epithelial cells (MAC-T) and human embryonic kidney 293 T cells (HEK293T) were used in our experiments. Dulbecco’s modified Eagle medium/Ham’s F-12 medium (DMEM-F12) (GIBCO, Cat. #1699748) was used as the basic medium for MAC-T, and Dulbecco’s modified Eagle medium (DMEM) (GIBCO, Cat. #1744353) was used as the basic medium for HEK293T. The growth medium comprised the basic medium supplemented with 500 U/L penicillin, 500 mg/L streptomycin (GIBCO, Cat. #15140163), and 10% (v/v) fetal bovine serum (GIBCO, Cat. #16000044). Both cells were maintained at 37 °C with 5% CO₂ and subcultured every other day.

MAC-T and HEK293T were used for transfection. The cells were cultured in six-well culture plates. One day before the transfection, the medium was replaced by removing the antibiotics. When the cells reached 80% confluence, 4 μg of the mini-gene reporters or pSPL3 vector was added to the Lipofectamine 2000 Transfection Reagent (Invitrogen, Cat. #11668030) in Opti-MEM medium (Gibco, Cat. #51985042). A growth medium without antibiotics was substituted for the Opti-MEM medium after 5 h. The cells were harvested 24 h after transfection, and total RNA was extracted with the RNAsimple Total RNA Kit (Tiangen, Cat. #DP419) to determine the expression of mini-gene reporters.

RNA extracted from the transfected cells was reverse transcribed into cDNA by using the pSPL3 vector-specific primer SA2 with the PrimeScript™ II First-strand cDNA Synthesis Kit (TaKaRa, Cat. #6210A). Specific primers for pSPL3 vector SD6 and SA2 were used to perform RT-PCR as previously described. The products were separated through electrophoresis on 1% agarose gels. Each DNA band was further analyzed by direct sequencing after extraction with the TIANgel Midi Purification Kit (Tiangen, Cat. #DP209).