[Complexity of Detecting CRISPR/Cas9-Mediated Homologous Recombination in Zebrafish].

Homology-directed (HD) genome modification offers an opportunity to precisely modify the genome. Despite reported successful cases, for many loci, precise genome editing remains challenging and inefficient in vivo. Here we report an effort to precisely knock-in a GFP reporter into gad locus mediated by CRISPR/Cas9 system in the zebrafish Danio rerio. PCR artifact was detected in testing for homologous recombination (HR), but was mitigated by optimizing PCR condition and decreasing the injected targeting plasmid concentration. Under this optimized condition, time course analysis revealed a decline of the HR-positive embryos at embryogenesis progressed. GFP signals also diminished at later developmental stages. The GFP signals were consistent with PCR detection, both of which suggested the loss of targeted insertion events at later stages. Such loss of insertion might be one underlying reason for the inability to obtain germ-line transgenic lines with GFP knocked into the gad locus. Our results suggest that the low HR efficiency associated with CRISPR-mediated knock-in is in part due to loss of insertion after targeted integration into the gad locus.

Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.

To investigate responses of milk protein synthesis and mammary AA metabolism to a graded decrease of postruminal Lys supply, 4 lactating goats fitted with jugular vein, mammary vein, and carotid artery catheters and transonic blood flow detectors on the external pudic artery were used in a 4 × 4 Latin square experiment. Goats were fasted for 24 h and then received a 9-h intravenous infusion of an AA mixture plus glucose. Milk yield was recorded and samples were taken in h 2 to 8 of the infusion period; a mammary biopsy was performed in the last hour. Treatments were graded decrease of lysine content in the infusate to 100 (complete), 60, 30, or 0% as in casein. Lysine-removed infusions linearly decreased milk yield, tended to decrease lactose yield, and tended to increase milk fat to protein ratio. Milk protein content and yield were linearly decreased by graded Lys deficiency. Mammary Lys uptake was concomitantly decreased, but linear regression analysis found no significant relationship between mammary Lys uptake and milk protein yield. Treatments had no effects on phosphorylation levels of the downstream proteins measured in the mammalian target or rapamycin pathway except for a tended quadratic effect on that of eukaryotic initiation factor 2, which was increased and then decreased by graded Lys deficiency. Removal of Lys from the infusate linearly increased circulating glucagon and glucose. Removal of Lys from the infusate linearly decreased arterial and venous concentrations of Lys. Treatments also had a significant quadratic effect on venous Lys, suggesting mechanisms to stabilize circulating Lys at a certain range. The 2 infusions partially removing Lys resulted in a similar 20% decrease, whereas the 0% Lys infusion resulted in an abrupt 70% decrease in mammary Lys uptake compared with that of the full-AA mixture infusion. Consistent with the abrupt decrease, mammary Lys uptake-to-output ratio decreased from 2.2 to 0.92, suggesting catabolism of Lys in the mammary gland could be completely prevented when the animal faced severe Lys deficiency. Mammary blood flow was linearly increased, consistent with the linearly increased circulating nitric oxide by graded Lys deficiency, indicating mechanisms to ensure the priority of the mammary gland in acquiring AA for milk protein synthesis. Infusions with Lys removed increased mammary clearance rate of Lys numerically by 2 to 3 fold. In conclusion, the decreased milk protein yield by graded Lys deficiency was mainly a result of the varied physiological status, as indicated by the elevated circulating glucagon and glucose, rather than a result of the decreased mammary Lys uptake or depressed signals in the mTOR pathway. Mechanisms of Lys deficiency to promote glucagon secretion and mammary blood flow and glucagon to depress milk protein synthesis need to be clarified by future studies.

Effect of dietary roughage level on chewing activity, ruminal pH, and saliva secretion in lactating Holstein cows.

Increasing dietary roughage level is a commonly used strategy to prevent subacute ruminal acidosis. We hypothesized that high-roughage diets could promote chewing activity, saliva secretion, and hence more alkaline to buffer rumen pH. To verify the hypothesis, 12 multiparous Holstein cows in mid lactation were randomly allocated to 4 treatments in a triplicated 4 × 4 Latin square experiment with one cow in each treatment surgically fitted with a ruminal cannula. Treatments were diets containing 40, 50, 60, or 70% of roughage on a DM basis. Increasing dietary roughage level decreased DM, CP, OM, starch, and NEL intake, increased ADF intake, and decreased milk yield linearly. Intake of NDF was quite stable across treatments and ranged from 7.8 to 8.1 kg/d per cow. Daily eating time increased linearly with increased roughage level. The increase in eating time was due to increased eating time per meal but not number of meals per day, which was stable and ranged from 8.3 to 8.5 meals per day across treatments. Increasing dietary roughage level had no effect on ruminating time (min/d), the number of ruminating periods (rumination periods per d), and chewing time per ruminating period (min/ruminating period). Ruminating time per kilogram of NDF intake and total chewing time per kilogram of ADF intake were similar across treatments (57.4 and 183.8 min/kg, respectively). Increasing dietary roughage level linearly increased daily total chewing time; linearly elevated the mean, maximum, and minimum ruminal pH; and linearly decreased total VFA concentration and molar proportion of propionate in ruminal fluid. Saliva secretion during eating was increased, the secretion during rumination was unaffected, but the secretion during resting tended to decrease with increased dietary roughage level. As a result, total saliva secretion was not affected by treatments. In conclusion, the results of the present study did not support the concept that high-roughage diets elevated ruminal pH through increased salivary recycling of buffering substrates.

Methionine, leucine, isoleucine, or threonine effects on mammary cell signaling and pup growth in lactating mice.

Two studies were undertaken to assess the effects of individual essential AA supplementation of a protein-deficient diet on lactational performance in mice using litter growth rates as a response variable. The first study was designed to establish a dietary protein response curve, and the second to determine the effects of Leu, Ile, Met, and Thr supplementation of a protein-deficient diet on lactational performance. In both studies, dams were fed test diets from parturition through d 17 of lactation, when the studies ended. Mammary tissue was collected on d 17 from mice on the second experiment and analyzed for mammalian target of rapamycin (mTOR) pathway signaling. Supplementation with Ile, Leu, or Met independently increased litter weight gain by 11, 9, and 10%, respectively, as compared with the protein-deficient diet. These responses were supported by independent phosphorylation responses for mTOR and eIF4E binding protein 1 (4eBP1). Supplementation of Ile, Leu, and Met increased phosphorylation of mTOR by 55, 34, and 47%, respectively, as compared with the protein-deficient diet. Phosphorylation of 4eBP1 increased in response to Ile and Met supplementation by 60 and 40%, respectively. Supplementation of Ile and Met increased phosphorylation of Akt/protein kinase B (Akt) by 41 and 59%, respectively. This work demonstrated that milk production responds nonlinearly to protein supply, and milk production and the mTOR pathway responded independently to supplementation of individual AA. The former demonstrates that a linear breakpoint model is an inappropriate description of the responses, and the latter demonstrates that no single factor limits AA for lactation. Incorporation of a multiple-limiting AA concept and nonlinear responses into milk protein response models will help improve milk yield predictions and allow derivation of diets that will increase postabsorptive N efficiency and reduce N excretion by lactating animals.

Distribution and localization of abnormally expressed OPTN proteins in RGC5 retinal ganglion cells and their effects on subcellular morphology.

The objectives of this study were to investigate the distributions of abnormally expressed optineurin (OPTN) proteins in retinal ganglion cells (RGC5s) of transgenic rats and their effects on subcellular morphological structures. Green fluorescent protein labeled EGFP wild-type (OPTN(WT)), E50K mutant type (OPTN(E50K)), and OPTN siRNA (si-OPTN) eukaryotic expression plasmids were constructed and transfected into RGC5s. Intracellular structures were labeled with organelle specific fluorescent dyes. Construct localization and cell morphologies were visualized by confocal fluorescence microscopy. OPTN(WT) was observed to be distributed as fine punctate fluorescent particles in the cytoplasm around the nucleus, along with exhibiting nuclear expression. OPTNE50K exhibited similar distribution but with non-uniform fluorescence particle size. si-OPTN distribution was similar to that of EGFP: uniform across the cytoplasm and nucleus. Compared with the negative control group, OPTN(WT), and OPTN(E50K) and to a lesser degree pEGFP-transfected cells exhibited fracture and loss of myofilament proteins and mitochondrial swelling and cytoplasmic accumulation, along with abnormal lysosomal distribution and increased volume, and Golgi fragmentation. However, si- OPTN transfected cells exhibited no significant damage. Therefore, we demonstrated that the E50K mutation disrupts the uniformity of OPTN protein distribution upon exogenous overexpression. Furthermore, these results suggested that si-OPTN transfection, and thus potentially OPTN knockdown, did not impact subcellular morphology of RGC5 cells, whereas transfection, especially when combined with wild-type or mutant OPTN expression, led to substantial abnormalities in subcellular morphological structures. These findings lay a foundation for further research into the function of the OPTN protein.

Effects of abnormal optineurin expression on the survival of the rat retinal ganglion cell line RGC-5.

The OPTN gene is thought to be associated with certain types of glaucoma and the function of the protein for which it encodes, optineurin, has been extensively researched, but with contradictory results. We explored the effects of abnormal optineurin expression on the survival of the rat retinal ganglion cell line RGC-5. Plasmids expressing wild-type (WT) or E50K mutant optineurin, or OPTN-specific double-hairpin small interfering RNA (si-RNA), were transfected into RGC-5 cells. The effects on cell survival were monitored by observation of cell morphology and propidium iodide and Hoechst 33342 fluorescent staining, while expression of optineurin was visualized by fluorescence microscopy. Abnormal optineurin expression influenced the survival of RGCs in vitro, as apoptosis was induced by increased WT and E50K mutant optineurin, while a reduction in apoptosis was observed in cells transfected with OPTN-siRNA. Similar results were also observed in transfected cells treated with apoptotic stimuli. Overexpression of WT and mutant E50K protein resulted in greater cell death, while downregulation decreased RGC-5 apoptosis.