Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology.
OBJECTIVE
To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia.


METHODS
Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown-multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Des1d yielding the final expression constructs pAV.Ex1d-cytomegalovirus (CMV)>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES /EGFP, respectively.


RESULTS
The adenovirus plasmids pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES/EGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells.


CONCLUSIONS
We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumican gene in the pathogenesis of high myopia.
Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China.
Gui-Fang
GF
Wang
Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China.
Bing
B
Qi
Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China.
Lei-Lei
LL
Tu
Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China.
Lian
L
Liu
Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China.
Guo-Cheng
GC
Yu
Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China.
Jing-Xiang
JX
Zhong
adenovirus vector; gateway recombinant cloning technology; lumican; myopia
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