title: Optimization of genetic engineering and homologous recombination of collagen type I genes in rat bone marrow mesenchymal stem cells (MSC).
abstract: Mutations in COL1A1 or COL1A2 genes lead to osteogenesis Imperfecta (OI) in humans. There are three possiblities to successfully treat OI including (1) gene therapy, (2) mesenchymal stem cell (MSC) therapy, or (3) a combination of both. The aim of this study was to develop a model for combined gene/cell OI therapy by targeting Col1a1 and Col1a2 genes with isogenic sequences from corresponding human genes in rat bone marrow (BM)-derived MSCs. The recombination efficacy was tested for five different rat-human-rat hybrid DNAs with rat fragments that were 1 to 4 kb long. For selection of transfected clones a neomycine resistance gene was cotransfected, and clones resistant to G418 (G418(+)) were recovered and screened for integration of specific gene loci in the rat genome. Over 90% of G418(+) clones correctly integrated the rat-human-rat hybrid DNAs, and both OI loci in the rat genome were targeted to a similar degree. Longer homologous sequences integrated into rat collagen genes approximately 10 times more efficiently. Based on our data the nonviral gene targeting technology could be potentially employed to repair collagen genes in OI patients.
authors.affiliation: Department of General and Molecular Biology and Genetics, Medical University of Silesia, Katowice, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Tarnowski
authors.forename: Anna
authors.initials: A
authors.lastname: Szydło
authors.forename: Jacek
authors.initials: J
authors.lastname: Anioł
authors.forename: Halina
authors.initials: H
authors.lastname: Koryciak-Komarska
authors.forename: Marta
authors.initials: M
authors.lastname: Lesiak
authors.forename: Ewa
authors.initials: E
authors.lastname: Gutmajster
authors.forename: Aleksander L
authors.initials: AL
authors.lastname: Sieroń
authors.forename: Damian
authors.initials: D
authors.lastname: Kusz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D024042:Collagen Type I / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers; D005818:Genetic Engineering; D006801:Humans; D006824:Hybridization, Genetic; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D011995:Recombination, Genetic; D014162:Transfection
chemical_list: D024042:Collagen Type I; D017931:DNA Primers