1. Volume 3, Issue 4, Pages 613-622 (April 2001)
A Cre-Expressing Cell Line and an E1/E2a Double-Deleted Virus for Preparation of Helper-Dependent Adenovirus Vector 
Heshan Zhou, Tiejun Zhao, Lucio Pastore, Maged Nageh, Wendy Zheng, X.Mei Rao, Arthur L. Beaudet 
Molecular Therapy 
Volume 3, Issue 4, Pages (April 2001)
DOI: /mthe
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
(A) Screening of the Cre-expressing cell clones by Cre/loxP deletion activation. The Cre/loxP activation mechanism is shown at the top. Plasmid pLoxZ contains two loxP sites that flank a DNA segment with a stop codon. Expression from the lacZ gene in a Cre-negative cell (E2T) is blocked because of premature termination. Excision of the segment between the loxP sites in Cre-expressing cells (E2T-Cre6) activates the expression of the lacZ gene. (B) Resistance to helper virus infection. 293, 293Cre4, E2T-Cre4, and E2T-Cre6 cells were infected with the helper virus AdLC8cluc at an m.o.i. of Efficient deletion of the viral packaging region in E2T-Cre6 cells prevented packaging of the viral DNA into infectious particles. The helper virus spread inefficiently in the E2T-Cre6 culture.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Southern blot assay for deletion of the packaging region in the helper virus DNA. Total DNAwas isolated from different cells infected with the Ad helper virus AdLC8cluc and cleaved with restriction enzyme PstI. (A) The probe containing the Ad ITR could hybridize with both Ad terminal fragments. Deletion of the packaging region in the helper virus decreased the leftterminal fragment from 0.53 to 0.26 kb. The size of the right terminal fragment did not change. The DNA isolated from Cre-negative 293 and E2T cells showed undeleted fragments, while the packaging region was deleted in the 293Cre4 cells. (B) The efficiency of the Cre/loxP-mediated deletion in different cell lines is shown.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Southern blot assay for purified HD vector DNA. (A) The probe DNA containing Ad ITR can hybridize with the terminal fragments of helper virus and HD vector DNA digested with the restriction enzyme PstI. (B) When the helper virus DNA was present, hybridization resulted in and 0.53-kb bands as labeled. The contamination of helper virus DNA was hardly detectable in the HD vector preparations.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Comparison of the titers of viruses with wild-type E2a or with deletion of E2a on 293, E2T, and E2T-Cre6 cells. Titer of the virus with E1 deleted, Adj8galAE1, is shown on the left, and titer of the virus with E1 and E2a deleted, AdβgalΔE1E2, is shown on the right. Viruses were collected from medium after 2 days of infection with an m.o.i. of 1, and the titer was measured as blue forming units (BFU) on E2T cells.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
PCR analysis of deletion in the packaging region in helper viruses. AdΔE1, AdLC8cluc, and AdhvΔE1E2 are E1-deleted viruses, while AdLC8cluc and AdhvDE1E2 contain two loxPs flanking the packaging region. The AdAEI without the inserted loxP was used as negative control; the AdLC8cluc containing two inserted loxP sites was used as positive control. PCR with AdΔE1 produced a 0.40-kb fragment regardless of the cell line. Insertion of two loxPs in AdLC8cluc and AdhvΔE1E2 increased the fragment size. However, deletion of the packaging region through loxP/Cre interaction in E2T-Cre6 cells produced shorter fragments.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions