Cloning and Fuctional Assessment of the Chicken Ovalbumin Promoter Sequences
Date Issued
2005
Date
2005
Author(s)
Huang, Chu-Ping
DOI
zh-TW
Abstract
A modern genetically selected White Leghorn hen lays up to 330 eggs per year and the total proteins contained within each egg has been averaged around 6.6 grams, including 3.5 grams of the egg-white proteins and 3.1grams of the yolk proteins. Of 3.5 grams of the egg-white proteins, 2 grams of them have been identified as the ovalbumin. The fact that egg-white proteins are primarily synthesized and secreted from the tubular gland cells lined on the lumen of the magnum of the oviduct, indicating that these tubular gland cells found in the magnum of laying chicken possess a great potency of serving as the bioreactor for production of transgenic proteins.
Attempts of the present studies were made to clone the promoter sequences of ovalbumin gene and to evaluate the potency of using the cloned promoter for construction of the transgene(s). To meet with the described purpose, genomic DNAs extracted from blood cells of the laying chicken hens were subjected to cloning of the ovalbumin promoter sequences by the strategy of PCR-TA cloning techniques that have resulted in a fragment of the genomic DNA lengthen in 3.1 kb was successfully amplified and ligated into the pGlow-TOPO vector. Of the 3.1 kb fragment after sequencing analysis, a 1.3 kb in length of the 5’ end sequences was confirmed to contain the promoter region and the rest fragment lengthen in 1.8 kb of the 3’ end was characterized as the sequences encoding the leading protein.
The efficacy of the cloned ovalbumin promoter was further evaluated using the green fluorescence protein (GFP) as a reporter. Of these studies, a transgene named pMAR-OV-GFP was constructed allowing the full length of GFP cDNA sequences driven by ovalbumin promoter and the OV-GFP sequences were equipped with a matrix attachment region (MAR) that will ensure the transgene successfully escaped from damage by those topoisomerases. The expression of transgene of pMAR-OV-GFP was evidenced from the observation of GFP fluorescence detected by fluorescent microscope around 24-48 post the transgene had been transfected into the primary cultured tubular gland cells of oviduct magnum from the laying hen. These results indicate that the cloned ovalbumin promoter does possess potency to drive the GFP cDNA while the efficiency is still far beyond satisfy.
Subjects
卵白蛋白啟動子
蛋白分泌部
管狀腺細胞
ovalbumin promoter
magnum
tubular gland cell
Type
thesis
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