RESUMO
Human embryonic stem cells (hESC) are considered to be an indefinite source of self-renewing cells that can differentiate into all types of cells of the human body and could be used in regenerative medicine, drug discovery and as a model for studying early developmental biology. hESC carrying disease-causing mutations hold promise as a tool to investigate mechanisms involved in the pathogenesis of the disease. In this report, we describe the behaviour of an expanded CTG repeat in the 3' untranslated region of the DMPK gene in VUB03_DM1, a hESC line carrying the myotonic dystrophy type 1 (DM1) mutation compared with the normal CTG repeat in two hESC lines VUB01 and VUB04_CF. Expanded CTG repeats were detected by small amount PCR, small pool PCR and Southern blot analysis in consecutive passages of VUB03_DM1. An important instability of the CTG repeat was detected during prolonged in vitro culture, showing stepwise increases of the repeat number in consecutive passages as well as a higher range of variability. This variability was present in cells of different colonies of the same passage and even within single colonies. The high repeat instability is in contrast to the previously observed stability of the repeat in preimplantation embryos and in fetuses during the first trimester of pregnancy. This in vitro culture of affected hESC represents a valuable model for studying the biology of repeat instability.
Assuntos
Células-Tronco Embrionárias/metabolismo , Instabilidade Genômica/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Expansão das Repetições de Trinucleotídeos/genética , Southern Blotting , Linhagem Celular , Células-Tronco Embrionárias/citologia , Humanos , Distrofia Miotônica/genética , Miotonina Proteína Quinase , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Human embryonic stem (hES) cells are pluripotent cells usually derived from the inner cell mass (ICM) of blastocysts. Because of their ability to differentiate into all three embryonic germ layers, hES cells represent an important material for studying developmental biology and cell replacement therapy. hES cell lines derived from blastocysts diagnosed as carrying a genetic disorder after PGD represent in vitro disease models. METHODS: ICMs isolated by immunosurgery from human blastocysts donated for research after IVF cycles and after PGD were plated in serum-free medium (except VUB01) on mouse feeder layers. RESULTS: Five hES cell lines were isolated, two from IVF embryos and three from PGD embryos. All lines behave similarly in culture and present a normal karyotype. The lines express all the markers considered characteristic of undifferentiated hES cells and were proven to be pluripotent both in vitro and in vivo (ongoing for VUB05_HD). CONCLUSIONS: We report here on the derivation of two hES cell lines presumed to be genetically normal (VUB01 and VUB02) and three hES cell lines carrying mutations for myotonic dystrophy type 1 (VUB03_DM1), cystic fibrosis (VUB04_CF) and Huntington disease (VUB05_HD).
Assuntos
Linhagem Celular , Embrião de Mamíferos/citologia , Fertilização in vitro , Doenças Genéticas Inatas/diagnóstico , Células-Tronco Pluripotentes/citologia , Diagnóstico Pré-Implantação , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular , Fibrose Cística/diagnóstico , Fibrose Cística/genética , Feminino , Humanos , Doença de Huntington/diagnóstico , Doença de Huntington/genética , Distrofia Miotônica/diagnóstico , Distrofia Miotônica/genética , GravidezRESUMO
A DNA complementary to the viral genome of C-type particles produced by a Mouse myeloma derived cell line (MF2 cell line) was synthesized. This cDNA was used as a probe to study the viral genome expression among the total RNA and the poly (A)-rich RNA extracted from the MF2 and Balb/c embryonic cells. As evidenced by molecular hybridization experiments, the presence of at least one endogenous Balb/c virus in the MF2 virus stocks is suggested. In the productive cells, the viral RNA sequences are expressed in the poly (A)-rich RNA fraction.
