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Knock! Knock! Where is there?
\"This hilarious follow-up collection of jokes featuring all the subjects of the ever-popular Where Is? series will keep kids laughing right through geography class! There are 300 silly jokes about places like Stonehenge, Easter Island, the Bermuda Triangle, Mount Rushmore, Area 51, and even our Solar System in this all-new collection\"-- Publisher's website.
Book
Visualization and Statistical Analysis of Passive Pre-chamber Knock in a Constant-volume Optical Engine
This study investigates the behavior of pre-chamber knock in comparison to traditional spark ignition engine knock, using a modified constant-volume gasoline engine with an optically accessible piston. The aim is to provide a deeper understanding of pre-chamber knock combustion and its potential for mitigating knock. Five passive pre-chambers with different nozzle diameters, volumes, and nozzle numbers were tested, and nitrogen dilution was varied from 0% to 10%. The stochastic nature of knock behavior necessitates the use of statistical methods, leading to the proposal of a high-frequency band-pass filter (37–43 kHz) as an alternative pre-chamber knock metric. Pre-chamber knock combustion was found to exhibit fewer strong knock cycles compared to SI engines, indicating its potential for mitigating knock intensity. High-speed images revealed pre-chamber knock primarily occurs near the liner, where end-gas knock is typically exhibited. The study identified that increasing pre-chamber nozzle diameter resulted in a larger dispersion of knock cycles and more severe knock intensity, likely due to shorter jet penetration depth requiring more time for end-gas consumption. Strategies for mitigating knock in pre-chamber combustion systems include reducing the pre-chamber volume for a fixed A/V ratio and increasing dilution level. The results of this study offer valuable insights for developing effective knock mitigation approaches in pre-chamber combustion systems, contributing to the advancement of more efficient and reliable engines.
Journal Article
Laugh-out-loud springtime jokes for kids
\"Rob Elliott's #1 bestselling Laugh-Out-Loud Jokes for Kids series returns with a collection of seasonal jokes to get you through the April showers. These bright, clean jokes and sunny puns are sure to freshen up the winter air. Get away from the screen and enjoy some laughs! Perfect for travels and for sharing with friends.\"--Provided by publisher.
Book
Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9
Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) mediated by microhomology-mediated end-joining, termed the PITCh (Precise Integration into Target Chromosome) system. TALEN-mediated PITCh, termed TAL-PITCh, enables efficient integration of exogenous donor DNA in human cells and animals, including silkworms and frogs. We further demonstrate that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence. Thus, our PITCh-ing strategies will be useful for a variety of applications, not only in cultured cells, but also in various organisms, including invertebrates and vertebrates.
One challenge facing the use of programmable nucleases in genome engineering is the requirement for homologous recombination. Here, Nakade
et al.
harness microhomology-mediated end-joining as a means of inserting exogenous coding sequences into the genome using both TALEN and CRISPR/Cas9 technologies.
Journal Article
Best kids' knock-knock jokes ever!
\"This sidesplitting humor collection features 365 uproarious knock-knock jokes--with tons of funny cartoons to complement the jokes. Kids will love amusing themselves--and friends and family--with this hilarious knock-knock collection\"--Provided by publisher.
Book
Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors
CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30-60% and reaches as high as 100% in some cases). The protocol takes â^¼2 months to generate the founder mice.
Journal Article
Knock! Knock! Who was there?
Over 300 side-splitting jokes based on the New York Times best-selling series. If you want to know exactly why Milton Hershey's wife married him, look no further. (Because she wanted lots of Hershey's Kisses!) This hilarious and original collection of jokes featuring all the subjects of the ever-popular Who Was? series will keep kids laughing right through history class!
Book
BS15 Ultra-highly flexible, accurate, high throughput platform for knock-in studies
BackgroundWhole genome screening with CRISPR targeted gene deletion has revolutionized research, but extension to knock in studies have been hampered reducing applicability and flexibility. Here, we describe a gene insertion high-throughput platform which greatly enhances possibilities of standard gene deletion screening technologies.MethodsThe CRISPR-CAS9 platform consists of a highly efficiently designed electroporator for (reversed) gene transfection, an ultra-high precision robot dispenser, and a modular parallel plate chamber for experimental interventions which are functionalized involving a series of optimized procedures. Analysis was performed by in house developed single cell analysis software operating on a high throughput microscope.ResultsA protocol was optimized to accurately deposit large numbers of small (<200 um) spots in shape, and position (Coefficient of Variation (CV)<10%). Effective RNAi loss-of-function is demonstrated for multiple gene targets at the mRNA (KLF-4), protein (cop-GFP) and phenotypic level with >70% transfection efficiency for hard-to-transfect endothelial cells, and ~100% transfection for tumor cell lines (HEK293T). Subsequent CRISPR/CAS9 mediated knock out directed against PRX27 in CAS9 +/+ fibroblasts showed a dose-dependent reduction in gene expression. Knock in studies in HEK293T-copGFP cells indicate a full recovery after gene deletion of GFP11 on gene level and protein level. Subsequent, large-scale knock-in studies confirm the low variability (<10%) and the capacity to create highly specific patterns in large scale (>10,000 spots) of protein-tagging.ConclusionThis study introduced a novel ultra-high precision high throughput platform with high scalability and excellent tunability for gene insertion experiments using knock in CRISPR. This platform greatly enhances functionalities of current CRISPR high throughput systems.Conflict of InterestNo
Journal Article
Laugh-out-loud A+ jokes for kids
A collection of school-related one-liners, knock-knock jokes, and tongue twisters.
Book
Telomerase reverse transcriptase gene knock‐in unleashes enhanced longevity and accelerated damage repair in mice
While previous research has demonstrated the therapeutic efficacy of telomerase reverse transcriptase (TERT) overexpression using adeno‐associated virus and cytomegalovirus vectors to combat aging, the broader implications of TERT germline gene editing on the mammalian genome, proteomic composition, phenotypes, lifespan extension, and damage repair remain largely unexplored. In this study, we elucidate the functional properties of transgenic mice carrying the Tert transgene, guided by precise gene targeting into the Rosa26 locus via embryonic stem (ES) cells under the control of the elongation factor 1α (EF1α) promoter. The Tert knock‐in (TertKI) mice harboring the EF1α‐Tert gene displayed elevated telomerase activity, elongated telomeres, and extended lifespan, with no spontaneous genotoxicity or carcinogenicity. The TertKI mice showed also enhanced wound healing, characterized by significantly increased expression of Fgf7, Vegf, and collagen. Additionally, TertKI mice exhibited robust resistance to the progression of colitis induced by dextran sodium sulfate (DSS), accompanied by reduced expression of disease‐deteriorating genes. These findings foreshadow the potential of TertKI as an extraordinary rejuvenation force, promising not only longevity but also rejuvenation in skin and intestinal aging. Tert knock‐in (TertKI) mice harboring the EF1α‐Tert gene in the Rosa26 locus displayed heightened telomerase activity, elongated telomeres, and extended lifespan. TertKI mice displayed enhanced wound healing and exhibited resistance to the progression of colitis.
Journal Article