Viable and fertile mice can be generated by somatic nuclear transfer into enucleated oocytes, presumably because the transplanted somatic cell genome becomes reprogrammed by factors in the oocyte. The first somatic cloned offspring of mice were obtained by directly injecting donor nuclei into recipient enucleated oocytes. When this method is used (the so-called Honolulu method of somatic cell nuclear transfer [SCNT]), the donor nuclei readily and completely condense within the enucleated metaphase II–arrested oocytes, which contain high levels of M-phase-promoting factor (MPF). It is believed that the condensation of the donor chromosomes promotes complete reprogramming of the donor genome within the mouse oocytes. Another key to the success of mouse cloning is the use of blunt micropipettes attached to a piezo impact-driving micromanipulation device. This system saves a significant amount of time during the micromanipulation of oocytes and thus minimizes the loss of oocyte viability in vitro. For example, a group of 20 oocytes can be enucleated within 10 min by an experienced operator. This protocol is composed of seven parts: (1) preparing micropipettes, (2) setting up the enucleation and injection micropipettes, (3) collecting and enucleating oocytes, (4) preparing nucleus donor cells, (5) injecting donor nuclei, (6) activating embryos and culturing, and (7) transferring cloned embryos.
Dengue is a still-growing public health concern in many tropical and subtropical regions of the world. The development and implementation of an effective dengue vaccine in these regions is a high priority. This insight focuses on the expected characteristics of a safe and efficacious vaccin ... more
Cell-free cytoplasmic extracts prepared from Xenopus eggs have been used extensively to recapitulate and characterize intracellular events in vitro. Egg extracts can be induced to transit the cell cycle and reconstitute assembly of dynamic structures including the interphase nucleus and the ... more
The circadian clock drives rhythms in the levels of thousands of proteins in the mammalian cell, arising in part from rhythmic transcriptional regulation of the genes that encode them. However, recent evidence has shown that posttranscriptional processes also play a major role in generating ... more