Improved Genome Editing via Oviductal Nucleic Acids Delivery (i-GONAD): Protocol Steps and Additional Notes.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice). Such experiments are performed by highly skilled technicians (especially for MI). We recently developed a novel genome editing method, called "GONAD (Genome-editing via Oviductal Nucleic Acids Delivery)," which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed "improved-GONAD (i-GONAD)." The i-GONAD method involves injection of CRISPR reagents into the oviduct of an anesthetized pregnant female using a mouthpiece-controlled glass micropipette under a dissecting microscope, followed by EP of the entire oviduct allowing the CRISPR reagents to enter into the zygotes present inside the oviduct, in situ. After the i-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. The i-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, the i-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about the i-GONAD method. Additionally, even though the detailed protocols of GONAD and i-GONAD have been published elsewhere (Gurumurthy et al., Curr Protoc Hum Genet 88:15.8.1-15.8.12, 2016 Nat Protoc 14:2452-2482, 2019), we provide all the protocol steps of i-GONAD in this chapter so that the reader can find most of the information, needed for performing i-GONAD experiments, in one place.

Indwelling catheters increase altered mental status and urinary tract infection risk: A retrospective Cohort Study.

BACKGROUND: Although indwelling urinary catheters (IUCs) are used intraoperatively and may cause complications (e.g., delirium), only few robust studies have investigated the association between intraoperative IUC use and complications. We hypothesized that IUC use might increase the postoperative incidence of altered mental status and/or urinary catheter infection. MATERIALS AND METHODS: In this retrospective single-center cohort study, we analyzed the data of adult patients undergoing surgery at our facility between January 2013 and December 2018. The primary endpoint was altered mental status and/or incidence of urinary catheter infections. The patients were divided into IUC and control groups. A multivariable logistic regression model was used to identify the predictors of postoperative complications, and a multivariable Cox proportional hazards regression model was used to analyze hospital discharge in unmatched and inverse propensity-weighted patients. RESULTS: Of the 14,284 patients that were reviewed, we analyzed 5112 patients (control group, 44.0%; IUC group, 56.0%). Almost all procedures comprised less invasive surgeries. The prevalence of postoperative altered mental status and postoperative urinary catheter infection were 3.56% and 0.04%, respectively. After inverse propensity weighting, all baseline characteristics were similar between the two groups. However, patients with IUCs had a higher risk of postoperative complications (adjusted odds ratio, 1.97; 95% confidence interval [CI], 1.50-2.59) and prolonged hospital stays (hazard ratio, 0.84; 95% CI, 0.80-0.89). CONCLUSION: In patients undergoing less invasive surgery, IUCs may be associated with a relatively high risk of altered mental status or urinary catheter infection. These data may facilitate preoperative discussions regarding the perioperative use of IUCs.