Pgl3 basic vector

Fragment primers for −1802, −1488, −1044, −708, −483, −216, −28 and +144 were designed (Table S1) to amplify unidirectional deletions of the bovine SIX1 promoter. Promoter constructs were generated by PCR using specific primers with the sequence of the KpnI and BglII restriction sites incorporated and the wild individuals as DNA templates. Then, all fragments were cloned into Vector pMD19-T (simple) (TaKaRa), and ligated into the luciferase reporter construct pGL3-basic vector digested with the same restriction enzymes KpnI and BglII (TaKaRa). These plasmids were named pGL3-1802, pGL3-1488, pGL3-1044, pGL3-708, pGL3-483, pGL3-216 and pGL3-28. The PCR amplification conditions were similar to the conditions used in the previous step and plasmid DNA was further confirmed by DNA sequencing.

Four fragments containing MC5R-5’ sequences from -1200 to -210 relative to the transcription initiation site were sub-cloned into pGL3-basic vector (Takara, China). Luciferase reporter assay procedure was performed as previously described [53 (link)]. Briefly, HEK293T cells were cultured in 24-well plates. Cells were co-transfected with Foxo4 overexpression vector, Renilla plasmid and pGL3-MC5R plasmid. pGL3-basic vector was considered as control reporter. Cells were harvested 48 h after transfection, and detected using the Dual-Luciferase Reporter assay system (Promega, USA). Luciferase activity was obtained by an average of luciferase assay experiments at least three times.

Four fragments containing Foxc2–5′ sequences from −1100 to −264 relative to the transcription initiation site were sub-cloned into pGL3-basic vector (Takara, China). HEK293T cells were cultured in 24-well plates and co-transfected with Foxc2 promoter plasmids and pGL3-basic plasmid (control reporter). Cells were harvested 48 h after transfection, and detected using the Dual-Luciferase Reporter assay system (Promega, USA). The MC5R promoter reporter assay and luciferase reporter assay were performed as the same protocol. And the four fragments containing MC5R-5′ sequences were from −1200 to −210 relative to the transcription initiation site.

The fragment primers SIX1-P1/R (−2000/+170), SIX1-P2/R (−1300/+170), SIX1-P3/R (−689/+170) and SIX1-P4/R (−40/+170) were designed to contain unidirectional deletions of the bovine SIX1 promoter. The PCR complexes were generated using specific primers that included the sequences of the KpnI and BglII restriction sites and the SIX1-PF/PR products as a template. PCR fragments were then cloned into the pMD19-T (simple) vector (Takara) and ligated into the luciferase reporter construct pGL3-basic vector digested with the same restriction enzymes KpnI and BglII (Takara). These plasmids were named pGL3-P1, pGL3-P2, pGL3-P3 and pGL3-P4.

Fragments containing TNC promoter sequences were subcloned into a pGL3-basic vector (Takara, China). HEK293T or 3T3-L1 cells were cultured in 24-well plates and co-transfected with TNC promoter plasmid and pc-Hoxa5 plasmid or pcDNA3.1 plasmid. After 48 h, then cells were harvested to analyze the activity using the Dual-Luciferase Reporter assay system (Promega, USA). The details were performed as previous addressed (22 (link)).

The ATF4 promoter sequence was analyzed using Genomatrix MatInspector. Two fragments containing ATF4–5′ sequences from –890 to –264 relative to the transcription initiation site were sub-cloned into pGL3-basic vector (Takara, Dalian, China). HEK293 cells were cultured in 24-well plates till 80–90% confluence and co-transfected with Renilla plasmid, pGL3-basic or PGL3-ATF4 plasmid (control reporter), and Smad3 overexpression plasmid (pc-Smad3). Cells were harvested 36 h after transfection and detected using the Dual-Luciferase Reporter assay system (Promega, Madison, WI, USA).

Four fragments containing Bim-5’ sequences were from -1200 to -210 relative to the transcription initiation site was sub-cloned into pGL3-basic vector (Takara, China), respectly. HEK293T cells were cultured in 24-well plates and co-transfected with Bim promoter plasmid and pc-Foxo1 plasmid or pGL3-basic plasmid (control reporter). Cells were harvested 48 h after transfection, and detected using the Dual-Luciferase Reporter assay system (Promega, USA). And luciferase activity was divided by all luciferase assay experiments were performed three times at least and each conducted in triplicate.
Chromatin immunoprecipitation (ChIP) assay was performed by using a ChIP assay kit (Abcam, Cambridge, UK) according to the manufacturer's protocol. DNA-protein crosslinking complexes were collected, and purified DNA was subjected to qPCR with SYBR green fluorescent dye (Invitrogen, Californian, USA).