Reprogramming and genetic modification of cells
A genetic modification, reprogramming technology, applied in the field of genetic defects, reprogramming and genetic modification, can solve problems such as low efficiency
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0090] Expression of Nanog protein in early embryos is required and associated with progressive X reactivation
[0091] In vivo reactivation of the X-chromosome is known to occur around 4.5dpc of the ectodermal mother cell of the female embryo (Mak et al., 2004; Okamoto et al., 2004). To investigate the putative involvement of Nanog proteins in reprogramming in vivo, we examined Eed and Nanog protein expression in 4.5dpc Nanog- / - embryos by immunofluorescence. Eed is a marker of preimplantation X chromosome inactivation (Silva et al., 2003; Mak et al., 2004; Okamoto et al., 2004). Of the 25 embryos produced by Nanog- / - mating, 6 lacked Nanog protein staining, and 4 of them were females. Nanog protein-negative embryos showed a large Eed focus in all nuclei, excluding apoptotic and poorly stained Eed cells ( Figure 1A ). Female Nanog protein-positive embryos showed varying numbers of Nanog protein-positive ICM cells (Fig. 1E). In contrast to the rest of this embryo, a propor...
Embodiment 2
[0094] Enhanced ability of ES cells overexpressing Nanog protein to generate hybrid colonies of ES-like ES-differentiated cells
[0095] To investigate whether the Nanog protein is involved in reprogramming, we used fusion cells of ES cells with other ES, NS, mouse embryonic fibroblasts (MEF) and thymocytes (T), and measured WT cells and cells manipulated by Nanog to produce ES Ability to hybridize colonies of ES-like differentiated cells. pre-generated using three different cell fusion methods, namely electrofusion, polyethylene glycol (PEG)-mediated fusion, and spontaneous fusion of cells during co-culture (Matveeva et al., 1998; Terada et al., 2002; Ying et al., 2002). Pluripotent hybrid cells. When we generated hybrid cells using PEG-induced fusion, we initially used RH ES cells constitutively expressing the red fluorescein and hygromycin resistance genes, and RH ES-derived cells RHN overexpressing the Nanog protein. Fusion of these cells with both ES and NS cells carryi...
Embodiment 3
[0101] PEG fails to induce RH cell death and increased differentiation compared with RH nanog ES cells
[0102] To assess whether PEG treatment might have a pro-differentiation effect on RH or RHN ES cells, we analyzed the survival and differentiation of the cells. Cell survival was scored as the number of ES cells attached to 100 mm plates 24 hours after PEG treatment. Vaccination 10 6 PEG-treated RH and RHN ES cells were counted to 6.6x10 the next day 5 and 5.3x10 5 cells ( Figure 3A ). To verify whether it is possible to induce differentiation and PEG-treated ES cell growth and alter the ability to form ES cell colonies, PEG-treated and untreated RH and RHN ES cells were subjected to single-cell FACS sorting. About 12 days after FACS sorting, the wells were stained with alkaline phosphatase (AP) for identification of undifferentiated ES cells. Most seeded untreated RH and RHN ES cells formed undifferentiated colonies ( Figure 3B , C), while PEG-treated cells showed...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More