1. Generation of transgenic mice using lentiviral vectors: a novel preclinical assessment of lentiviral vectors for gene therapy 
Masahito Ikawa, Nobushige Tanaka, Winston W.-Y Kao, Inder M Verma 
Molecular Therapy 
Volume 8, Issue 4, Pages (October 2003)
DOI: /S (03)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Preparation of oncoretroviral (RV) and lentiviral vectors (LV). (A) Diagram of lentiviral (top) and retroviral (bottom) vectors used for transduction of EGFP. All oncoretroviral and lentiviral vectors carry a posttranscriptional regulatory element of the woodchuck hepatitis virus (wPRE) [14]. Lentiviral vectors carry self-inactivating deletion mutations (open triangle) [10] and a central polypurine tract (cPPT) [11] of HIV-1 and each kind of promoter (Pr) to drive EGFP ubiquitously or in a tissue-specific manner. BamHI site (arrow) is a unique site in both lentiviral and retroviral vectors. RSV, promoter sequence from respiratory syncytial virus; Ψ, packaging signal. The approximate sizes of various promoters used are shown. (B) Transduction of 293T cells by oncoretroviral (right) and lentiviral (left) vectors expressing EGFP under the control of the CMV promoter. The cells were observed 2 days after the transduction at an m.o.i. of 1.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Generation of transgenic mice using oncoretroviral and lentiviral vectors. (A) Transgenic pups derived from LV-CAG-EGFP- (upper row) or RV-CAG-EGFP- (lower row) treated embryo. Green fluorescence was observed under a fluorescence stereomicroscope (right of each row) and photos were taken under similar conditions. The whole body of the LV-CAG-EGFP transgenic pup is also shown (bottom). (B) The copy numbers of transgenic founder mice generated by LV-CMV-EGFP or RV-CMV-EGFP treatment were determined (top) by Southern blot analysis. Each specific integration band is indicated (white arrowhead). Genomic DNA from each mouse was digested with BamHI and hybridized with an EGFP probe. Lane 1, 10 pg of GFP-LV; lane 2, genomic DNA from wild-type mouse. EGFP protein expression in muscle tissue of those mice was also examined (bottom) by Western blot analysis. Lane 1, positive control, 1 μg of protein derived from muscle tissue of LV-CAG-EGFP transgenic mouse; lane 2, negative control, 10 μg of protein derived from muscle tissue of wild-type mouse. (C) The inheritance of transgenes to the F1 progeny was confirmed in the transgenic founder generated by LV-CMV-EGFP (top). Each specific integration band indicated by each small letter in lane 3 (F0-1) was also detected in the F1 progeny. EGFP protein expression in muscle tissue of those mice was also examined (bottom) by Western blot analysis. Lanes 1 and 2, see (B). (D) EGFP expression in each tissue of wild-type mouse (top), transgenic mice generated by LV-CAG-EGFP (second row) and RV-CAG-EGFP (third row) treatment, and transgenic mouse generated by pronuclear injection of plasmid carrying EGFP cassette driven by CAG promoter (TgN(beta-act-EGFP)) (bottom) was examined by Western blot analysis. Ten or 1 μg of protein derived from each tissue of wild-type and RV-CAG-EGFP transgenic mouse or LV-CAG-EGFP transgenic mouse and TgN(beta-act-EGFP) was loaded into each well. Proteins transferred to the membrane were probed by anti-EGFP antibody. One and 10 μg of protein derived from muscle tissue of transgenic mouse generated by LV-CAG-EGFP were used as positive controls (P1 and P10, respectively).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Tissue-specific expression with lentiviral vectors: photoreceptor cells. (A) Transverse sections of eyes derived from wild-type mouse (left) and LV-Rho-EGFP transgenic mouse (right) were observed by light (top) or fluorescence (bottom) microscope. (B) The eye section of an LV-Rho-EGFP transgenic mouse was further examined with higher magnification. EGFP (top), DAPI (middle), and overlaid (bottom) images are shown. Only the cells located at the outer nuclear layer (ONL), where the photoreceptor cells exist, expressed EGFP. (INL, inner nuclear layer.) (C) The eye section of an LV-RG-EGFP transgenic mouse was examined. EGFP (top), DAPI (middle), and overlaid (bottom) images are shown. Only the cells confined to the outer half of the ONL, where the cone photoreceptor cells exist, expressed EGFP. Autofluorescence derived from the interphotoreceptor matrix was detected. (D) EGFP expression in each tissue from an LV-Rho-EGFP transgenic mouse (top) and an LV-RG-EGFP transgenic mouse (bottom) was examined by Western blot analysis. Ten micrograms of protein derived from each tissue was loaded into each well. One and 10 μg of protein derived from muscle tissue of a transgenic mouse generated by LV-CAG-EGFP were used as positive controls (P1 and P10, respectively).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Tissue-specific expression with lentiviral vectors: cornea. (A) Transverse sections of eyes derived from wild-type mouse (left) and LV-K12-EGFP transgenic mouse (right) were observed by light (top) or fluorescence (bottom) microscope. The area surrounded by a square was examined with higher magnification in (B). Bright-field (upper left), EGFP (upper right), DAPI (lower left), and overlaid (lower right) images are shown. Only the corneal epithelial cells expressed EGFP. Arrows indicate limbus, which is the border area between the corneal and the conjunctival epithelial cells. (C) EGFP expression in each tissue of an LV-K12-EGFP transgenic mouse (top and middle) and endogenous keratin-12 expression in each tissue of a wild-type mouse (bottom) was examined by Western blot analysis. Ten micrograms of protein derived from each tissue was loaded into each well. 1 and 10 μg of protein derived from the muscle tissue of an LV-CAG-EGFP transgenic mouse were used as positive controls (P1 and P10, respectively).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions