Targeted Gene Therapy for Ischemic Stroke - Gene Delivery and Therapy for Neurological Disorders - page 195

4. Solvents: xylene, graded ethanol (70 %, 90 %, 95 %, 100 %)
5. Cover slips
6. Permount
7. NIH ImageJ 1.63 software ( http://rsb.info.nih.gov/ij )

3 Methods

3.1 Hypoxia-
Inducible Gene
Expression AAV Vector

Hypoxia-inducible gene expression is based on using HRE, and
HRE sequences are found in gene enhancers that are inducible by
hypoxia. Here, we used Epo HRE as an example. Nine copies of
Epo HRE consensus sequences [ 63 , 64 ] (Fig. 1a ) were cloned
between Sma I and Nhe I sites of p gal-control vector (Clontech)

before the SV40 minimum promoter. Different copies of Epo HRE
and HREs isolated from other genes can also be cloned to the same
position ( see Note 1 ). The capacity of AAV vector is limited to
4.5 kb. The polyadenylation signal in p

ʲ

ʲ gal-control vector is about

a

9 copies of Epo HRE consensus sequence:

CTGCAGGAATTCGATGCACGCGTCCGGGTAGCTGGCG TACGTG CTGCAGCCGGGTAGCT

GGCG TACGTG CTGCAGCCGGGTAGCTGGCG TACGTG CTGAGCTCGAGACTTGACGCGTC

CGGGTAGCTGGCG TACGTG CTGCAGCCGGGTAGCTGGCG TACGTG CTGCAGCCGGGTAG

CTGGCG TACGTG CTGAGCTCGAGACTTGACGCGTCCGGGTAGCTGGCG TACGTG CTGCA

GCCGGGTAGCTGGCG TACGTG CTGCAGCCGGGTAGCTGGCG TACGTG CTGCAGCTCGAG

b

H9LacZ

9HRE PmSV40 LacZ PA

Sma I Nhe I Sal I

VEGF

H9VEGF

9HRE PmSV40 VEGF PA

Sma I Sal I

Fig. 1 ( a ) Nine copies of Epo HRE consensus sequence. The HIF-1 binding sites are underlined . ( b ) Schematic
drawing of H9LacZ and H9VEGF. Nine copies of Epo HRE (9HRE) were cloned in front of the minimum SV40
promoter (PmSV40). H9VEGF was generated by replacing the LacZ sequence between Hind III and Nde I with
human VEGF 165 cDNA