Submitted to: Gene
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 1, 2005
Publication Date: June 1, 2005
Citation: Zheng, J., Ather, J.L., Sonstegard, T.S., Kerr, D.E. 2005. Characterization of the infection-responsive bovine lactoferrin promoter. Gene. 353:107-17.
Interpretive Summary: Understanding the control mechanisms of a potential transgene promoter is a critical step in designing disease resistant transgenic animals where exogenous gene expression is properly coordinated both temporally and spatially during an infection within an animal. This study was directed at understanding the promoter and enhancer elements that direct expression of bovine lactoferrin transcripts. First, the promoter region of bovine lactoferrin was isolated and sequenced using DNA from a bovine artificial chromosome. Lactoferrin was determined to be expressed throughout the body, but most expression is in the mammary gland and liver. The ability of Escherichia coli lipopolysaccharidde (LPS; a common antigen that triggers immune response during infection) to induce reporter gene expression under control of the bovine lactoferrin promoter was detected. Next, this upstream region containing LPS inducible control elements was truncated in varying lengths and the ability of each resulting deletion to induce expression of a downstream reporter gene was determined. These results showed the promoter region motifs (NfkB binding site) were responsible for induction of transcription through LPS stimulation. This research lays the framework for alteration and use of the bovine lactoferrin promoter as a promoter to control expression of antibiotic transgenes in the mammary gland to fight bacterial infections causing mastitis.
The concentration of lactoferrin in bovine milk is dramatically increased in response to infection. The high levels of lactoferrin may have a role in the prevention of microbial infection of the mammary gland. However, molecular mechanisms of how the lactoferrin gene is regulated in the mammary gland in response to infection remain unknown. In this study, we isolated and characterized the 5' flanking region of the bovine lactoferrin gene. An 8.2 kilobase (kb) fragment of the bovine lactoferrin gene, containing 4.4-kb of 5' flanking region, exon 1, intron 1 and exon 2, was isolated from a bovine genomic library on two overlapping bacterial artificial chromosome (BAC) clones. Sequence analysis of the isolated lactoferrin gene revealed that the promoter region contains a high GC content, a noncanonical TATA box, and multiple stimulating protein 1 (SP1)/GC elements, and other putative binding sites for transcription factors including nuclear factor kB (NF-kB), activator protein 1 (AP1), signal transduction and activator of transcripton 3 and 5 (STAT3 and STAT5), and steroid hormone receptors. To demonstrate that the isolated promoter is functional, 4.4-kb of 5' flanking region was inserted upstream from the firefly luciferase gene and the chimeric construct was transiently transfected into murine mammary epithelial cells. Transfection studies showed that the basic promoter activity is quite potent, being similar in strength to that of the simian virus 40 (SV40) promoter/enhancer. In addition, a 24-hour treatment with Escherichia coli lipopolysaccharidde (LPS) significantly stimulated its activity up to 2.3 fold in a dose-dependent manner. Furthermore, promoter deletion analysis indicated that the sequence up to '543 was sufficient for basal activity, whereas the sequence up to '1029 was required for maximal basal activity. The basal activity of the promoter is affected by both positive regulatory regions (-2462/-1879 and -1029/-75) and a negative regulatory region (-1407/-1029). LPS-responsive regions of the promoter were localized to the region from '1029 to -543 containing one STAT3 site and two NF-kB sites, and the region from '4355 to '2462 containing three AP1 sites and six NF-kB sites. Taken together, our findings suggested that the lactoferrin promoter responds to infection via the NF-kB pathway.