Neurospheres were mechanically dissociated into single cells and transiently transfected with 4 μg of pCMV6-Entry vector carrying full length human HB-EGF plasmids (Origene, Rockville, MD) or with scrambled DNA control plasmid, using an Amaxa Basic Nucleofector kit (Lonza, Allendale, NJ) and the Amaxa Nucleofactor apparatus (Lonza, Allendale, NJ). Transfection efficiency was determined by real-time PCR using the following human HB-EGF primers: sense: 5′-CTCTCCCTGCCAAGTCTCAG -3′ and anti-sense: 5′-CTGCATGGAGTAGCACCAGA -3′. Transfected NSC were incubated in DMEM/F12 medium supplemented with 5% fetal bovine serum for 24h prior to administration in vivo.
Pcmv6 entry vector carrying full length human hb egf plasmids
Manufactured by OriGene
The PCMV6-Entry vector carrying full length human HB-EGF plasmids is a laboratory tool used to express the human heparin-binding EGF-like growth factor (HB-EGF) protein in cell lines. The vector contains the cytomegalovirus (CMV) promoter to drive the expression of the full-length HB-EGF gene.
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2 protocols using pcmv6 entry vector carrying full length human hb egf plasmids
1
Transfection of Neurospheres with HB-EGF
2
Transfection of Neurospheres with HB-EGF
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Neurospheres were mechanically dissociated into single cells and transiently transfected with 4 μg of pCMV6-Entry vector carrying full length human HB-EGF plasmids (Origene, Rockville, MD) or with scrambled DNA control plasmid, using an Amaxa Basic Nucleofector kit (Lonza, Allendale, NJ) and the Amaxa Nucleofactor apparatus (Lonza, Allendale, NJ). Transfection efficiency was determined by real-time PCR using the following human HB-EGF primers: sense: 5′-CTCTCCCTGCCAAGTCTCAG -3′ and anti-sense: 5′-CTGCATGGAGTAGCACCAGA -3′. Transfected NSC were incubated in DMEM/F12 medium supplemented with 5% fetal bovine serum for 24h prior to administration in vivo.
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