Analysis of gene transfer and expression in skeletal muscle using enhanced EIAV lentivirus vectors  J.P O'Rourke, H Hiraragi, K Urban, M Patel, J.C Olsen, B.A Bunnell  Molecular Therapy  Volume 7, Issue 5, Pages 632-639 (May 2003) DOI: 10.1016/S1525-0016(03)00074-1 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Lentivirus gene transfer and gene expression in proliferating and differentiated C2C12 cells. (A) Cells were transduced with the various vector systems at an m.o.i. of 5 (proliferating) or 10 (differentiated) and analyzed for gene transfer 7 days posttransduction by 25 cycles of PCR using EGFP or β-actin-specific primers. The blot shown is representative of four independent experiments. (B) Cells transduced as above were analyzed for EGFP expression by flow cytometry 7 days posttransduction. The results are average percentages of EGFP-positive cells ± standard deviation of four independent experiments. Molecular Therapy 2003 7, 632-639DOI: (10.1016/S1525-0016(03)00074-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Lentivirus gene transfer into skeletal muscle after intramuscular injections. Mice were sacrificed at 1 week and 1 and 3 months after vector administration and analyzed for gene transfer levels by semiquantitative PCR. (A) 35 cycles of PCR on DNA isolated from 3-month animals using EGFP- or β-actin-specific primers. (B) Average levels of gene transfer into skeletal muscle from all animals analyzed by PCR. Data are presented as percentage nuclei containing vector sequence. PCR was performed at various cycles to ensure PCR signal was in the linear range. Molecular Therapy 2003 7, 632-639DOI: (10.1016/S1525-0016(03)00074-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 EGFP mRNA expression from lentivirus vectors in skeletal muscle after intramuscular injections. Mice were sacrificed at 1 week and 1 and 3 months after vector administration and analyzed for EGFP mRNA levels by RT-PCR. (A) 40 cycles of RT-PCR on total RNA isolated from 3-month animals using EGFP- or β-actin-specific primers. (B) Average densitometry levels of EGFP mRNA in skeletal muscle from all animals analyzed by RT-PCR. (C) Average densitometry levels of EGFP mRNA in skeletal muscle from all animals analyzed by RT-PCR normalized to vector copies per nuclei. Data are presented as arbitrary densitometry units from each RT-PCR band divided by the corresponding levels of vector copy number from the same animal. RT-PCR was performed at various cycles to ensure PCR signal was in the linear range. Molecular Therapy 2003 7, 632-639DOI: (10.1016/S1525-0016(03)00074-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Immunohistochemistry detection of EGFP protein in skeletal muscle. (A) Skeletal muscle from mock- or EIAV-injected mice 1 week and 1 and 3 months post-vector administration was isolated, formalin fixed, sectioned, and analyzed for EGFP protein using an EGFP-specific antibody followed by Alexa Fluor 568 secondary antibody. Staining in muscle fibers of EIAV-injected animals is shown at all time points. (B) Confocal microscopic analysis of immunohistochemically stained fibers. Bright-field analysis shows Z bands characteristic of myocytes. Staining is shown in myocyte cytoplasm of 1-month EIAV-injected animal. Molecular Therapy 2003 7, 632-639DOI: (10.1016/S1525-0016(03)00074-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Comparison of first- and second-generation EIAV vectors in skeletal muscle. Mice were injected with 1 × 106 infectious particles of EIAV (first generation) or E-SIN vectors. Mice were sacrificed 1 week later and DNA and RNA were isolated from skeletal muscle for analysis of gene transfer by PCR and EGFP mRNA levels by RT-PCR. (A) DNA PCR from 1-week animals. (B) RT-PCR from 1-week animals. (C) Graphic representation of DNA and RT-PCR densitometry data from EIAV and E-SIN vectors. Error bars represent standard deviation. Molecular Therapy 2003 7, 632-639DOI: (10.1016/S1525-0016(03)00074-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions