Floxing by Electroporating Single-Cell Embryos with Two CRISPR RNPs and Two ssODNs.

Floxed alleles and Cre drivers are two components of most conditional knockout mouse models, which are not only important for studying a given gene in a tissue-specific manner, but also useful for functional analysis of various sized genomic regions. With the increased demand for floxed mouse models in biomedical research, reliable and economical creation of floxed alleles is clearly highly valuable yet remains challenging. Here we provide technical details on the method consisting of electroporating single-cell embryos with CRISPR RNPs and ssODNs, next-generation sequencing (NGS)-based genotyping, an in vitro Cre assay (recombination followed by PCR) for loxP phasing determination, and optional second round targeting of an indel in cis with one loxP insertion in embryos obtained via in vitro fertilization (IVF). As importantly, we present protocols for validation of gRNAs and ssODNs before electroporation of embryos, to confirm phasing of loxP and the indel to be retargeted in individual blastocysts and an alternative strategy to insert loxP sites sequentially. Together, we hope to help researchers reliably obtain floxed alleles in a predictable and timely manner.

Effect of tea polyphenols on the oral and intravenous pharmacokinetics of ticagrelor in rats and its in vitro metabolism.

Green tea is widely consumed as a beverage and/or dietary supplement worldwide, resulting in the difficulty to avoid the comedication with ticagrelor for acute coronary syndrome (ACS) patients receiving antiplatelet therapy. This study was designed to investigate the effect of the most abundant content in green tea, tea polyphenols on the oral and intravenous pharmacokinetics of ticagrelor in rats and its in vitro metabolism. Rats were orally treated with either saline or tea polyphenol extracts (TPEs) dissolved in saline once daily for 6 consecutive days. On day 6, after the last dose of saline or TPE, ticagrelor was given to the rats orally or intravenously. Plasma samples were collected for pharmacokinetic analysis. Human liver and intestinal microsomes were then used to investigate the inhibition by TPE, as well as its major constituents on the metabolism of ticagrelor to its two metabolites, AR-C124910XX and AR-C133913XX. Apparent kinetic constants and inhibition potency (IC50 ) for each metabolic pathway of each compound were estimated. Oral study indicated that exposure of ticagrelor and AR-C124910XX was significantly decreased after TPE administration, while no significant differences were observed in pharmacokinetic parameters after intravenous administration of ticagrelor. TPE effectively inhibited the metabolism of ticagrelor in vitro, with epigallocatechin-3-gallate as the major constituent responsible for the observed inhibitory effects in human liver microsomes and intestinal microsomes (IC50 = 4.23 ± 0.18 µM). Caution should be taken for ACS patients receiving ticagrelor therapy with daily drinking of green tea. PRACTICAL APPLICATION: Potential interactions between tea polyphenols and ticagrelor were revealed for the first time. Results can provide suggestions for clinicians to optimize the dosing of ticagrelor while they are in the face of ACS patients receiving ticagrelor therapy, who also take green tea or its related products in their daily life.

AAH2 gene is not required for dopamine-dependent neurochemical and behavioral abnormalities produced by Toxoplasma infection in mouse.

Infection with the protozoan parasite, Toxoplasma gondii (T. gondii), has been associated with the increased risk for several psychiatric disorders. The exact mechanisms of a hypothesized contribution of T. gondii infection are poorly understood. The T. gondii genome contains two aromatic amino acid hydroxylase genes (AAH1 and AAH2) that encode proteins that can produce L-DOPA. One popular hypothesis posits that these encoded enzymes might influence dopamine (DA) production and hence DA synaptic transmission, leading to neurobehavioral abnormalities in the infected host. Prior studies have shown that deletion of these genes does not alter DA levels in the brain or exploratory activity in infected mice. However, possible effects of AAH gene deficiency on infection-induced brain and behavior alterations that are directly linked to DA synaptic transmission have not been evaluated. We found that chronic T. gondii infection of BALB/c mice leads to blunted response to amphetamine or cocaine and decreased expression of Dopamine Transporter (DAT) and Vesicular Monoamine Transporter 2 (VMAT2). Deletion of AAH2 had no effects on these changes in infected mice. Both wild type and Δaah2 strains produced comparable levels of neuroinflammation. Our findings demonstrate that AAH2 is not required for T. gondii infection-produced DA-dependent neurobehavioral abnormalities.

Structural correlates of cytoplasmic and chloroplast lipid body synthesis in Chlamydomonas reinhardtii and stimulation of lipid body production with acetate boost.

Light microscopy and deep-etch electron microscopy were used to visualize triacylglyceride (TAG)-filled lipid bodies (LBs) of the green eukaryotic soil alga Chlamydomonas reinhardtii, a model organism for biodiesel production. Cells growing in nitrogen-replete media contain small cytoplasmic lipid bodies (α-cyto-LBs) and small chloroplast plastoglobules. When starved for N, ß-cyto-LB formation is massively stimulated. ß-Cyto-LBs are intimately associated with both the endoplasmic reticulum membrane and the outer membrane of the chloroplast envelope, suggesting a model for the active participation of both organelles in ß-cyto-LB biosynthesis and packaging. When sta6 mutant cells, blocked in starch biosynthesis, are N starved, they produce ß-cyto-LBs and also chloroplast LBs (cpst-LBs) that are at least 10 times larger than plastoglobules and eventually engorge the chloroplast stroma. Production of ß-cyto-LBs and cpst-LBs under the conditions we used is dependent on exogenous 20 mM acetate. We propose that the greater TAG yields reported for N-starved sta6 cells can be attributed to the strain's ability to produce cpst-LBs, a capacity that is lost when the mutant is complemented by a STA6 transgene. Provision of a 20 mM acetate "boost" during N starvation generates sta6 cells that become so engorged with LBs-at the expense of cytoplasm and most organelles-that they float on water even when centrifuged. This property could be a desirable feature for algal harvesting during biodiesel production.

Algal lipid bodies: stress induction, purification, and biochemical characterization in wild-type and starchless Chlamydomonas reinhardtii.

When the unicellular green soil alga Chlamydomonas reinhardtii is deprived of nitrogen after entering stationary phase in liquid culture, the cells produce abundant cytoplasmic lipid bodies (LBs), as well as abundant starch, via a pathway that accompanies a regulated autophagy program. After 48 h of N starvation in the presence of acetate, the wild-type LB content has increased 15-fold. When starch biosynthesis is blocked in the sta6 mutant, the LB content increases 30-fold, demonstrating that genetic manipulation can enhance LB production. The use of cell wall-less strains permitted development of a rapid "popped-cell" microscopic assay to quantitate the LB content per cell and permitted gentle cell breakage and LB isolation. The highly purified LBs contain 90% triacylglycerol (TAG) and 10% free fatty acids (FFA). The fatty acids associated with the TAGs are approximately 50% saturated (C(16) and C(18)) fatty acids and approximately 50% unsaturated fatty acids, half of which are in the form of oleic acid (C(18:1)). The FFA are approximately 50% C(16) and approximately 50% C(18). The LB-derived TAG yield from a liter of sta6 cells at 10(7) cells/ml after starvation for 48 h is calculated to approach 400 mg. The LB fraction also contains low levels of charged glycerolipids, with the same profile as whole-cell charged glycerolipids, that presumably form LB membranes; chloroplast-specific neutral glycerolipids (galactolipids) are absent. Very low levels of protein are also present, but all matrix-assisted laser desorption ionization-identified species are apparent contaminants. Nitrogen stress-induced LB production in C. reinhardtii has the hallmarks of a discrete pathway that should be amenable to additional genetic and culture condition manipulation.