Construction of eukaryotic expression vector of short hairpin RNA targeting human xylosyltransferase-I gene / 华西口腔医学杂志
West China Journal of Stomatology
;
(6): 206-210, 2008.
Article
in Chinese
| WPRIM
| ID: wpr-296676
ABSTRACT
<p><b>OBJECTIVE</b>To design and construct the plasmids expressing short hairpin RNA (shRNA) targeting human xylosyltransferase- I (XT- I) which is the initiating enzyme in the biosynthesis of proteoglycans (PC).</p><p><b>METHODS</b>Short chain oligonucleotides were designed according to the sequence of XT-I provided by GenBank. The DNA segments were gained through annealing after chemosynthesis, and were cloned into Pgenesil-1 vector. The recombinant XT- I shRNA expression vectors were identified by digestion and sequencing analysis. At last the constructed XT-I expression vectors were transfected into salivary adenoid cystic carcinoma cell line (ACC-M) by Lipofectomine 2000. The expression of green fluorescent protein (GFP) was detected by inverted fluorescent microscope and the rates of transfection were detected by flow cytometer. Semiquantitative RT-PCR was used to detect the expression of mRNA level of XT- I in transfected ACC-M cells and the protein expression of XT- I was detected by Western blot.</p><p><b>RESULTS</b>The plasmids expressing shRNA targeting XT-I gene are called WJ1, WJ2, WJ3, WJ4, WJ5 and WJ6. Successful constructions were identified by digestion and sequencing. The mean rate of transfection was 50.26%. ACC-M cells transfected with WJ1-WJ6 expressed GFP successfully. And by RT-PCR and Western blot, WJ3 showed the most powerful RNAi gene silencing of inhibitory. The inhibition rate was 72.39% of mRNA level and 70.18% of protein level respectively.</p><p><b>CONCLUSION</b>The XT-I shRNA expression vectors were constructed successfully which lays the foundation for RNAi study on the biosynthesis of PG in salivary gland tumors.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Pentosyltransferases
/
Plasmids
/
RNA, Messenger
/
Salivary Gland Neoplasms
/
Transfection
/
Cell Line
/
Carcinoma, Adenoid Cystic
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RNA, Small Interfering
/
RNA Interference
/
Green Fluorescent Proteins
Limits:
Humans
Language:
Chinese
Journal:
West China Journal of Stomatology
Year:
2008
Type:
Article
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