Volume 10, Issue 4, Pages 679-687 (October 2004) Nucleofection of muscle-derived stem cells and myoblasts with ϕC31 integrase: stable expression of a full-length-dystrophin fusion gene by human myoblasts  Simon P. Quenneville, Pierre Chapdelaine, Joël Rousseau, Jean Beaulieu, Nicolas J. Caron, Daniel Skuk, Philippe Mills, Eric C. Olivares, Michele P. Calos, Jacques P. Tremblay  Molecular Therapy  Volume 10, Issue 4, Pages 679-687 (October 2004) DOI: 10.1016/j.ymthe.2004.05.034 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Schematic representation of the three plasmids used in the present study. On the top, plasmid pCR3.1eGFP contains mainly the cDNA coding for eGFP under the control of the CMV promoter. In the middle, plasmid pCR3.1attBeGFP is the same as the previous plasmid, except that the attB sequence has been introduced in front of the CMV promoter. In the lower schema, plasmid pDysE [14] has been modified by insertion of the attB fragment in front of the CMV promoter. Note that the graphic representations of the constructs are not in scale. Molecular Therapy 2004 10, 679-687DOI: (10.1016/j.ymthe.2004.05.034) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Expression of eGFP by MD1 and human myoblasts in culture after nucleofection with pCMVInt. (A–C) eGFP expression in MD1 cells (upper picture) 48 h after nucleofection with, respectively, pCR3.1eGFP, pCR3.1attBeGFP, and pCR3.1attBeGFP + pCMVInt. (D–F) eGFP expression in human myoblasts (lower picture) 72 h after nucleofection with, respectively, pCR3.1eGFP, pCR3.1attBeGFP, and pCR3.1attBeGFP + pCMVInt. Note that the upper picture corresponding to each fluorescent image represents identical fields visualized by phase contrast. Original magnification ×20. Molecular Therapy 2004 10, 679-687DOI: (10.1016/j.ymthe.2004.05.034) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Stable expression of eGFP after co-nucleofection with pCR3.1attBeGFP and pCMVInt. Cells underwent selection with geneticin and culture for 6 weeks. (A) MD1 cells expressing eGFP (lower picture) and corresponding phase contrast (upper image). (B) Human myoblast clones stably expressing eGFP (lower picture) with the corresponding image showing phase contrast (upper image). Original magnification for all panels was ×20. Molecular Therapy 2004 10, 679-687DOI: (10.1016/j.ymthe.2004.05.034) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Expression of eGFP and eGFP–full-length dystrophin. Expression of the fusion protein, eGFP–full-length dystrophin (A–D, and F), and eGFP (E) in human myoblasts and myotubes after nucleofection or co-nucleofection with pCMVInt. Phase contrast (upper) and fluorescence (lower) pictures of the human myoblasts in primary culture 48 h after nucleofection with pCR3.1attBDysE (A) and (B) shows fluorescent granules within the cells. (C, D) eGFPDysE expression in cells, respectively, 8 and 10 days after co-nucleofection with pCMVInt (i.e., respectively after 5 and 7 days of geneticin selection); (E) Two different areas of fluorescent myotubes formed by the fusion of selected myoblasts 1 month after their co-nucleofection with pCR3.1attBeGFP and pCMVInt and geneticin selection (7 days). (F) Two different of areas of fluorescent myotubes obtained by the fusion of human myoblasts 1 month after the co-nucleofection of the myoblasts with pCR3.1attBDysE and pCMVInt followed by 7 days of geneticin selection. Molecular Therapy 2004 10, 679-687DOI: (10.1016/j.ymthe.2004.05.034) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Western blot analysis of eGFP-full-length dystrophin (A) and of full-length dystrophin (B). The proteins are originating from myotubes formed by the fusion of human myoblasts co-nucleofected with pCR3.1attBeGFP or pCR3.1attBDysE with pCMVInt. (A) A western blot using an antibody specific for eGFP reacting with proteins extracted from different parts of the culture containing fluorescent myotubes formed by the fusion of myoblasts co-nucleofected with pCR3.1attBeGFP and pCMVInt (lane 1), or co-nucleofected with pCR3.1attBDysE and pCMVInt (lanes 2, 3, and 4). Lanes 2 and 4 represent proteins extracted from fluorescent regions, whereas lane 3 represents proteins extracted from nonfluorescent cells. (B) A western blot done with the NCL-Dys2 antibody; all samples are in the same order as in (A). All lanes show an intense signal for the presence of full-length dystrophin (427 kDa) in the myotubes, but lane 1 shows only one band, whereas double bands of dystrophin were observed in lanes 2 and 4. This doublet results from the expression of both normal full-length dystrophin (427 kDa) and eGFP–full-length dystrophin (450 kDa). Molecular Therapy 2004 10, 679-687DOI: (10.1016/j.ymthe.2004.05.034) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Detection of integrase-mediated site-specific recombination in genomic DNA of human myoblasts stably expressing eGFP. Comparison of PCR products between human myoblasts non-nucleofected (lanes 1 and 2) and co-nucleofected with pCR3.1 attBeGFP and pCMVInt (lanes 4 and 5). [AUTHOR: d. LABELS CONFLICT WITH PREVIOUS SENTENCE. PLEASE CORRECT ONE OR THE OTHER TO BE CONSISTENT.] Lanes 1 and 4 show PCR products of dystrophin exon 45 (∼176 bp) as a genomic DNA control. Lanes 2 and 5 represent PCR products with one specific primer for the psA site (chromosome 8) and another specific primer for the attB sequence resulting in the expected PCR product (∼400 bp) for lane 5 only. Lane 3 shows a 1-kb DNA ladder on a 1.8% agarose gel. Molecular Therapy 2004 10, 679-687DOI: (10.1016/j.ymthe.2004.05.034) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions