Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination.

Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2's pathophysiological roles for the first time in a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem using CRISPR-Cas9 to edit embryos is mosaicism; thus, we studied for the first time the effect of microinjection timing on mosaicism. Mosaicism was greatly reduced when in vitro produced embryos were microinjected before insemination, and surgical embryo transfer (ET) was performed using such embryos. All TPC2 KO fetuses and piglets born following ET (i.e., F0 generation) were nonmosaic biallelic KOs. The generation of nonmosaic animals greatly facilitates germ line transmission of the mutation, thereby aiding the rapid and efficient generation of KO animal lines for medical research and agriculture.

PLCζ is the physiological trigger of the Ca2+ oscillations that induce embryogenesis in mammals but conception can occur in its absence.

Activation of the egg by the sperm is the first, vital stage of embryogenesis. The sperm protein PLCζ has been proposed as the physiological agent that triggers the Ca2+ oscillations that normally initiate embryogenesis. Consistent with this, recombinant PLCζ induces Ca2+ oscillations in eggs and debilitating mutations in the PLCZ1 gene are associated with infertility in men. However, there has been no evidence that knockout of the gene encoding PLCζ abolishes the ability of sperm to induce Ca2+ oscillations in eggs. Here, we show that sperm derived from Plcz1-/- male mice fail to trigger Ca2+ oscillations in eggs, cause polyspermy and thus demonstrate that PLCζ is the physiological trigger of these Ca2+ oscillations. Remarkably, some eggs fertilized by PLCζ-null sperm can develop, albeit at greatly reduced efficiency, and after a significant time-delay. In addition, Plcz1-/- males are subfertile but not sterile, suggesting that in the absence of PLCζ, spontaneous egg activation can eventually occur via an alternative route. This is the first demonstration that in vivo fertilization without the normal physiological trigger of egg activation can result in offspring. PLCζ-null sperm now make it possible to resolve long-standing questions in fertilization biology, and to test the efficacy and safety of procedures used to treat human infertility.

Calcium signals regulated by NAADP and two-pore channels--their role in development, differentiation and cancer.

Ca(2+) signals regulate a wide range of physiological processes. Intracellular Ca(2+) stores can be mobilized in response to extracellular stimuli via a range of signal transduction mechanisms, often involving recruitment of diffusible second messenger molecules. The Ca(2+) mobilizing messengers InsP 3 and cADPR release Ca(2+) from the endoplasmic reticulum via InsP 3 and ryanodine receptors, respectively, while a third messenger, NAADP, releases Ca(2+) from acidic endosomes and lysosomes. Bidirectional communication between the ER and acidic organelles has functional relevance for endolysosomal function as well as for the generation of Ca(2+) signals. The two-pore channels (TPCs) are currently strong candidates for being key components of NAADP-regulated Ca(2+) channels. Ca(2+) signals have been shown to play important roles in embryonic development and cell differentiation; however, much remains to be established about the exact signalling mechanisms involved. Investigation of the role of NAADP and TPCs in development and differentiation is still at an early stage, but recent studies have suggested that they play important roles at key developmental stages in vivo and are important mediators of differentiation of neurons, skeletal muscle cells and osteoclasts in vitro. NAADP signals and TPCs have also been implicated in autophagy, an important process in differentiation. Moreover, potential links between TPC2 and cancer have been recently identified. Further studies will be required to identify the precise mechanisms of action of TPCs and their link with NAADP signalling, and to relate these to their roles in differentiation and other key developmental processes in the cell and organism.