AIM: 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. CONCLUSION: 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.
AIM: 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. CONCLUSION: 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.
Authors: Marja Majava; Paul N Bishop; Pasi Hägg; Paul G Scott; Aine Rice; Chris Inglehearn; Christopher J Hammond; Tim D Spector; Leena Ala-Kokko; Minna Männikkö Journal: Hum Mutat Date: 2007-04 Impact factor: 4.878
Authors: Katie M Williams; Geir Bertelsen; Phillippa Cumberland; Christian Wolfram; Virginie J M Verhoeven; Eleftherios Anastasopoulos; Gabriëlle H S Buitendijk; Audrey Cougnard-Grégoire; Catherine Creuzot-Garcher; Maja Gran Erke; Ruth Hogg; René Höhn; Pirro Hysi; Anthony P Khawaja; Jean-François Korobelnik; Janina Ried; Johannes R Vingerling; Alain Bron; Jean-François Dartigues; Astrid Fletcher; Albert Hofman; Robert W A M Kuijpers; Robert N Luben; Konrad Oxele; Fotis Topouzis; Therese von Hanno; Alireza Mirshahi; Paul J Foster; Cornelia M van Duijn; Norbert Pfeiffer; Cécile Delcourt; Caroline C W Klaver; Jugnoo Rahi; Christopher J Hammond Journal: Ophthalmology Date: 2015-05-13 Impact factor: 12.079