To assess the promoter activity of the
M-CSF gene, we generated luciferase reporter constructs containing wild-type and mutant
M-CSF promoters based on a single dual-luciferase reporter plasmid pFRL2 [
24]. The
M-CSF promoter region (from −1983 to +1 bp relative to the transcription start site) was obtained from the genomic DNA of 293 T cells using the polymerase chain reaction (PCR) with the primer pairs 5′-TACACAGCAAATGAATGGCAGAGCTGG-3′ (forward) and 5′-GCGTCTTCCTAGTCACCCTCTGTCTTCTG-3′ (reverse), and cloned into the TA cloning vector yT&A, excised by digestion with
SmaI/
BglII, and cloned into the
SmaI/
BglII sites of pFRL2 to generate pFRL2
-M-CSF(−1983 ~ +1). We also constructed four deletion mutants in the
M-CSF promoter region encompassing −1063 ~ +1, −903 ~ +1, −577 ~ +1, and −487 ~ +1 bp (from the transcription start site). The promoter regions of the four deletion mutants were obtained from pFRL2-M-CSF(−1983 ~ +1) by PCR with the forward primers, including (−1063) 5′-ACTGC
ACGCGTATGAGCCAAGTCCA-3′, (−903) 5′-TCTGC
ACGCGTCAGTCTGAGCAAAG-3′, (−577) 5′-CATGG
ACGCGTTTCCAATCTGAGTTG-3′, and (−487) 5′-TAAGG
ACGCGTTGAAGTGTCTGCTGG-3′, as well as the reverse primer 5′-TATAT
CTCGAGCACCCTCTGTCTTCTGCG-3′. The PCR products were then ligated into the yT&A vector. These promoter regions with various deletions were then excised from the TA vector by digestion with
XhoI/
MluI (underlined) and cloned into the
XhoI/
MluI sites of pFRL2 to generate pFRL2-M-CSF(−1063 ~ +1), pFRL2-M-CSF(−903 ~ +1), pFRL2-M-CSF(−577 ~ +1), and pFRL2-M-CSF(−487 ~ +1). In addition, three luciferase reporter vectors containing the
M-CSF promoter carrying a point mutation (A
TGC
AATT ➔ A
CGC
GATT) at −980 bp within the first Oct4 response element (ORE1) site, a point mutation (
ATG
CA
AAT ➔
CTG
AA
GAT) at −530 bp within the second ORE site (ORE2), and a double mutation within both ORE1 and ORE2 sites were generated using pFRL2-M-CSF(−1983 ~ +1) as the template by site-directed mutagenesis by overlap extension using PCR [
25]. The primers (point mutation underlined) used include mutant 1, 5′-GAGA
CGC
GATTTCAGCCTGAAATGATGAGGAGTT-3′ (forward) and 5′-CTGAAAT
CG
CGTCTCATCCTCCACCAGCAAAGC-3′ (reverse); mutant 2, 5′-GCAT
CT
TCA
GCATCTAAGGGTCAGGTGCCTTGAA-3′ (forward) and 5′-TG
CTG
AA
GATGCTGGCTGGTACCCATGCT-3′ (reverse); and pFRL2, 5′-CCAGCCCAAGCTACCATGATAAGTAAG-3′ (forward) and 5′-CTTATGCAGTTGCTCTCCAGCGG-3′ (reverse). Finally, two outer primers 5′-TACACAGCAAATGAATGGCAGAGCTGG-3′ (forward) and 5′-GCGTCTTCCTAGTCACCCTCTGTCTTCTG-3′ (reverse) were used to synthesize the entire DNA sequence by PCR. The PCR products were cloned into the yT&A vector. The resulting constructs were then digested with
EcoRI/
BglII, and the released mutant fragments were cloned into the
EcoRI/
BglII sites of pFRL2 to generate pFRL2-M-CSF-ORE1mut (designated Mut1), pFRL2-M-CSF-ORE2mut (designated Mut2), and pFRL2-M-CSF-ORE1mut-ORE2mut (designated Dmut). All the cloned fragments were sequenced to validate their correctness.