The CRISPR/Cas9 lentiviral system has emerged as a powerful tool for gene knockout in cancer immunology research. However, the persistent expression of exogenous elements, such as Cas9 and resistance markers, often triggers excessive tumor immune rejection. This can lead to prolonged experimental timelines, increased data variability, biased outcomes, and even experimental failures. To address this challenge, several strategies have been investigated that offer partial solutions, but they have failed so far to comprehensively resolve the issue. In response, we developed the VL-AdCre system, a strategy that enables efficient excision of exogenous expression elements following gene knockout. The VL-AdCre system effectively reduced tumor immune rejection in allograft models, streamlined experimental workflows, and improved the reliability of research outcomes. Overall, this optimized CRISPR/Cas9 lentiviral gene knockout system offers a robust and practical solution for studying gene functions in vivo and advancing immunotherapeutic strategies.
To determine furaltadone metabolites 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ) residual levels in ctenopharyngodon idellus, direct competitive enzyme-linked immunosorbent assay (dcELISA) was developed based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein. Firstly, VH and VL gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against AMOZ derivative, and then the productive VH and VL genes were assembled to a scFv gene and engineered to produce scFv-AP fusion protein. The fusion protein was further identified as a bifunctional reagent for immunoassay, then a sensitive one-step dcELISA against AMOZ derivative was developed with a half-maximal inhibitory concentration (IC50) and limit of detection (LOD) of 10.62 and 4.83 ng/mL, respectively. The recovery values of AMOZ from spiked fish samples determined by dcELISA were in 71.31%∼82.88%. The results suggested that the prepared scFv-AP fusion protein in this work can be used for rapid and convenient immunoassays to detect AMOZ residues in fish samples.
Yang Chen,Jie Zhang,Wenhao Xiao,Anfu Chen,Zhixian Dong,Jinbao Xu,Wenhua Xu,Caihong Lei
1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) was used as organocatalyst to reinvestigate the ring-opening polymerization (ROP) of ε -caprolactone (CL) with benzyl alcohol (BnOH) initiator in bulk at 90 °C considering the unsuccessful ROP of CL at RT in previously reported publications. Kinetic study indicates that the PCLs could be well-designed with a controlled/living character of polymerization. The use of functional initiators, such as 2-hydroxyethyl methacrylate (HEMA), propargyl alcohol (PGA), 6-azido-1-hexanol (AHA) and methoxy poly(ethylene glycol) (mPEG) leads to end-functionalized PCLs. Accordingly, the block copolymerization of CL with δ-valerolactone (VL), trimethylene carbonate (TMC) and lactide (LA) successfully proceeded to give PCL- b -PVL, PCL- b -PTMC and PCL- b -PLA copolymers. Finally, the reaction mechanism was studied with DFT calculations and NMR measurements.
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