title: Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors.
abstract: Human and mouse embryonic stem (ES) cells have the capacity to differentiate into derivatives of all three germ layers, suggesting novel therapies for degenerative, metabolic, and traumatic disorders. ES-based regenerative medicine will be further advanced by the development of reliable methods for transgene introduction and expression. Here, we show infection of human and mouse embryonic stem (ES) cells with two of the most popular vectors in gene transfer, adenovirus type 5 (Ad5) and adeno-associated virus (AAV; serotypes 2, 4, and 5). All vectors express the nuclear-localized marker gene beta-galactosidase expressed from the Rous Sarcoma Virus long terminal repeat (RSV-LTR). Both Ad5 and AAV2 infected human and mouse ES cells and gave rise to beta-galactosidase expression. AAV4 and 5 did not yield detectable levels of beta-galactosidase expression. Quantitative PCR analysis of virally infected human and mouse ES cells revealed that only Ad5 and AAV2 are capable of transducing both cell-types. No viral DNA was detected in cells infected with either AAV4 or AAV5. Infection and subsequent differentiation of mouse and human ES cells with Ad5 showed that beta-galactosidase-expressing cells were restricted to cells in the interior of the embryoid body mass. No beta-galactosidase expression was observed in AAV-infected cells following differentiation. There was no difference in morphology or differentiation patterns between infected and noninfected differentiating mouse and human ES cells. Differentiation of hES cells prior to infection led to transduction of neuronally differentiated cells with good efficiency using all vectors. These data show that Ad5- and AAV2-based vectors are capable of infecting both human and mouse ES cells, in both their undifferentiated and differentiated states, whereas AAV4 and AAV5 can infect human and mouse ES cells only following differentiation.
authors.affiliation: Department of Gene Expression and Development, Roslin Institute, Roslin, Midlothian, United Kingdom.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Smith-Arica
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Thomson
authors.forename: Ray
authors.initials: R
authors.lastname: Ansell
authors.forename: John
authors.initials: J
authors.lastname: Chiorini
authors.forename: Beverly
authors.initials: B
authors.lastname: Davidson
authors.forename: Jim
authors.initials: J
authors.lastname: McWhir
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002454:Cell Differentiation; D000229:Dependovirus / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology*; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D016133:Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D001616:beta-Galactosidase