Journal: BioMed Research International
Article Title: Nonviral Gene Targeting at rDNA Locus of Human Mesenchymal Stem Cells
Figure Lengend Snippet: Site-specific integration at the rDNA locus of MSCs. (a) Schematic of the construction of pHr2-NL. pHr2-NL contained two inverted expression cassettes, one consisting of an IRES element from the encephalomyocarditis virus, the coding region of the Neo gene, the SV40 polyA signal (SV40pA), and two loxP sites with the same orientation. LoxP sites were recognized by CRE enzyme to remove the Neo cassette after gene targeting. LHA, long homologous arm (U13369:937-6523); SHA, short homologous arm (U13369:6523–7643). The genomic locus indicates the 6.7 kb fragment (U13369:937-7643) required for homologous recombination at the internal transcribed spacer 1 (ITS1) of the rRNA gene. Single cutting sites for restricted enzymes of Nco I, EcoR I, Hind III , and Pvu II are located at the IRES-Neo frame and outside of the long homologous fragment. The fragment between the two Pvu II sites was 8285 bp in size, and it was detected using probe 1 (P1). The expected sizes of the restriction fragments produced by Nco I, EcoR I, and Hind III were 4001 bp, 7628 bp, and 15,316 bp, respectively. These were detected using probe 2 (P2). Primer t-up was located at the SV40 polyA. Primer t-re was located outside of the SHA at the hrDNA locus. (b) Drug-resistant cell in basal medium. (c) Drug-resistant colonies in the medium supplemented with VEGF+bFGF+Vc+ITS-X. (d) Identification of colonies with site-specific integration by PCR. The expected fragment, 1.3 kb in size, was amplified from the genomic DNA of colonies using site-specific integration. M, DL200 DNA marker; 1, negative colony; 2–5, positive colonies; 6, wild-type MSCs. (e–f) Southern blotting analysis of the representative recombinants. Genomic DNA digested with Pvu II , Nco I, EcoR I, and Hind III was analyzed. A specific band was consistently detected in colonies 1-1, 1-2, 2-1, and 2-2. An additional band beside the specific band was detected in colony 2-3. c, control (untransfected MSCs); N, Nco I; E, EcoR I; H, Hind III .
Article Snippet: Southern Blotting After overnight digestion with restriction enzymes Pvu II, Nco I, EcoR I, and Hind III (New England Biolabs, Ipswich, MA, USA), 3 μ g of genomic DNA per sample was electrophoresed on a 0.8% agarose gel overnight and then transferred to positively charged nylon membranes (Hybond-N+, Amersham, Piscataway, NJ, USA).
Techniques: Expressing, Homologous Recombination, Produced, Polymerase Chain Reaction, Amplification, Marker, Southern Blot