Stem Cell Genetic Modification Study Method

Introducing Mutations Into Stem Cells

During the past decades, various approaches to the genetic modification of embryos have been conducted to determine the direct effects on organismal development. In the 1970s, the first genetic modification experiment was conducted in the mouse, which involved the introduction of the S40 virus to murine embryos that were at the blastocyst stage. Monitoring these embryos showed that the virus faithfully integrated into the genome of the mouse embryo, which in turn could continuously be traced to the live-born mouse. Other infectious viruses have also been tested afterward, including the Moloney murine leukemia virus that also resulted in effective genome integration. Subsequent years witnessed the introduction of exogenous genes into one-cell stage embryos through microinjection. Over time, the approaches in integrating various amount and kinds of genetic material were conducted, with almost all experiments generating highly informative results.

Mutational analysis of stem cells is an essential component in investigating the processes involved in self-renewal and differentiation of pluripotent stem cells. This approach also assists in identifying particular developmental disorders that could ensue in the presence of specific modification in the genome. Using the mouse as a convenient and highly information animal model, hundreds of mutations have now been characterized. This information has then been further applied to human embryonic stem cells as well as induced pluripotent stem cells. However, there are yet some genes that are quite difficult to characterize in stem cells; these include those that are duplicated and occur in segmented regions of the genome. It is actually not surprising that these segmented duplications remain major challenges to both geneticists and developmental biologists. Over time, it is expected that these recent changes in the human genome would be better understood once more sensitive approaches to genetic analysis are developed.

One approach in introducing mutations to a stem cell would be through insertional mutagenesis, which is generally conducted through the microinjection of fragments of DNA into embryos. It is also possible to perform insertional mutagenesis through retroviral infection. Previous studies have shown that insertional mutagenesis results in the random integration of DNA segments into the genome of the target stem cell. This, in turn, results in the identification of different types of genes that show a propensity for [Page 1074]integration and potentially an effect on the development of the embryo. There are currently hundreds of insertional mutations that have been identified and associated with embryonic development. The introduction of retroviruses for insertional mutagenesis also facilitates in the generation of chimeras that could be subsequently characterized using various genetic approaches such as expression profiling, DNA sequencing, and high-throughput characterization schemes such as mass spectrometry.

...