Derivation Of Hes Cell Lines Harboring Specific Genetic Defects

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The ability of hES cells to differentiate into each cell type of the adult body may be used for research on the nature and course of specific diseases. Models established by the use of hES cell lines carrying specific genetic defects may be highly effective in the development of drug or gene therapy designed to treat these diseases.

Two methods of obtaining such lines are (1) genetic manipulation of existing hES cell lines and (2) derivation of hES cell lines from genetically compromised embryos. Since the chance that donated surplus embryos from the in vitro fertilization (IVF) program carry genetic diseases is relatively low, the preferable source of donated embryos would be non-retrieved embryos from the preimplantation genetic diagnosis (PGD) program. PGD is designed for couples who are carriers of genetic diseases to ensure the transfer of healthy embryos to the uterus by their examination prior to implantation. For this purpose, the embryos are grown in vitro to the 6-8 cell stage, at which point one or two cells are removed and analyzed either by polymerase chain reaction (PCR) or by fluorescence in situ hybridization (FISH).

In our experience, post-PGD embryos continue to develop in vitro to the blastocyst stage. Five hES cell lines that were isolated from donated post-PGD blastocysts were derived and found to possess hES cell features. One line was found to harbor the Van Waardenburg disease (deletion at the PAX3 gene), and the other had myotonic dystrophy. The cell lines carrying genetic diseases could therefore be used for the development of in vitro models for these disorders.

Derivation of hES Cell Subclones hES cell lines are derived from the ICM, which may not represent a homogeneous cell population. To eliminate the possibility that the pluripotency of the lines reflects a collection of several distinct committed multipotential cell types in the culture, parental lines have to be single-cell cloned. The main aim of the first derivation of hES single-cell clones was to establish the pluripotency of the parental lines, but as with mES cells, single-cell cloning has additional advantages.

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