A simple and effective genotyping workflow for rapid detection of CRISPR genome editing.

Genetically engineered mouse models play a pivotal role in the modeling of diseases, exploration of gene functions, and the development of novel therapies. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing technology has revolutionized the process of developing such models by enabling precise genome modifications of the multiple interested genes simultaneously. Following genome editing, an efficient genotyping methodology is crucial for subsequent characterization. However, current genotyping methods are laborious, time-consuming, and costly. Here, using targeting the mouse trypsinogen genes as an example, we introduced common applications of CRISPR-Cas9 editing and a streamlined cost-effective genotyping workflow for CRISPR-edited mouse models, in which Sanger sequencing is required only at the initial steps. In the F0 mice, we focused on identifying the presence of positive editing by PCR followed by Sanger sequencing without the need to know the exact sequences, simplifying the initial screening. In the F1 mice, Sanger sequencing and algorithms decoding were used to identify the precise editing. Once the edited sequence was established, a simple and effective genotyping strategy was established to distinguish homozygous and heterozygous status by PCR from tail DNA. The genotyping workflow applies to deletions as small as one nucleotide, multiple-gene knockout, and knockin studies. This simplified, efficient, and cost-effective genotyping shall be instructive to new investigators who are unfamiliar with characterizing CRISPR-Cas9-edited mouse strains.NEW & NOTEWORTHY This study presents a streamlined, cost-effective genotyping workflow for clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) edited mouse models, focusing on trypsinogen genes. It simplifies initial F0 mouse screening using PCR and Sanger sequencing without needing exact sequences. For F1 mice, precise editing is identified through Sanger sequencing and algorithm decoding. The workflow includes a novel PCR strategy for distinguishing homozygous and heterozygous statuses in subsequent generations, effective for small deletions, multiple-gene knockouts, and knockins.

Multifragmentary patellar fracture has a distinct fracture pattern which makes coronal split, inferior pole, or satellite fragments.

The present study aimed to map the location and frequency of fracture lines on the coronal articular and sagittal planes in multifragmentary patellar fractures. 66 multifragmentary patellar fractures were digitally reconstructed using the 3D CT mapping technique. The coronal articular surface and midsagittal fracture maps were produced by superimposing each case over a single template. Each fracture line was classified based on the initial displacement and orientation. We evaluated the frequency and direction of the fracture line, coronal split fragment area, and satellite and inferior pole fragment presence. Coronal articular surface fracture mapping identified primary horizontal fracture lines between the middle and inferior one-third of the articular surface in 63 patients (95.4%). Secondary horizontal fracture lines running on the inferior border of the articular facet were confirmed (83.3%). Secondary vertical fracture lines creating satellite fragments were mostly located on the periphery of the bilateral facet. Midsagittal fracture mapping of primary and secondary horizontal fracture lines with the main coronal fracture line revealed a predominantly X-shaped fracture map. The consequent coronal split fragment and inferior pole fracture were combined in most cases. In conclusion, the multifragmentary patellar fracture has a distinct pattern which makes coronal split, inferior pole, or satellite fragments.

TRAF4 is a critical molecule for Akt activation in lung cancer.

TRAF4 is an adapter protein overexpressed in certain cancers, but its contributions to tumorigenesis are unclear. In lung cancer cells and primary lung tumors, we found that TRAF4 is overexpressed. RNA interference-mediated attenuation of TRAF4 expression blunted the malignant phenotype in this setting, exerting inhibitory effects on cell proliferation, anchorage-independent growth, and tumor development in a xenograft mouse model. Unexpectedly, we discovered that TRAF4, but not Skp2, was required for activation of the pivotal cell survival kinase Akt through ubiquitination. Furthermore, TRAF4 attenuation impaired glucose metabolism by inhibiting expression of Glut1 and HK2 mediated by the Akt pathway. Overall, our work suggests that TRAF4 offers a candidate molecular target for lung cancer prevention and therapy.