[Construction of eukaryotic expression vector of short hairpin RNA targeting human xylosyltransferase-I gene].
OBJECTIVE
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).


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Shi
Hong
H
Department of Oral Pathology, College of Stomatology, Hebei Medical University, Shijiazhuang 050017, China.
Wang
Jie
J
Wang
Xu
X
Gu
Hong-Tao
HT
Hou
Ya-Li
YL
Yu
Li-Jie
LJ
D003528:Carcinoma, Adenoid Cystic; D002460:Cell Line; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D010430:Pentosyltransferases; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012468:Salivary Gland Neoplasms; D014162:Transfection; D000097608:UDP Xylose-Protein Xylosyltransferase
D049452:Green Fluorescent Proteins; D010430:Pentosyltransferases; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000097608:UDP Xylose-Protein Xylosyltransferase