为了实现精子的内源性分离,本研究利用CRISPR/Cas9技术,通过基因双链断裂后的同源修复机制,将睾丸特异性启动子SP-10和干扰精子发育重要基因SPAG6、PPP1CC的shRNA偶联后定点整合进宁乡猪肾成纤维细胞X染色体的ZFX基因,构建并验证基因定...为了实现精子的内源性分离,本研究利用CRISPR/Cas9技术,通过基因双链断裂后的同源修复机制,将睾丸特异性启动子SP-10和干扰精子发育重要基因SPAG6、PPP1CC的shRNA偶联后定点整合进宁乡猪肾成纤维细胞X染色体的ZFX基因,构建并验证基因定点整合的细胞系。通过构建2个递送载体,根据人工多顺反子-tRNA-gRNA(PTG策略)基因串联表达系统原理,靶向ZFX基因设计多对sgRNA,验证sgRNA剪切效率,并构建sgRNA双顺反子切割载体。基于同源修复机制和同源介导末端连接(Homology Mediated End Joining,HMEJ)策略,构建含有新霉素抗性、EGFP标记基因等筛选标记的外源基因敲入载体,细胞电穿孔共转染后通过新霉素抗性筛选在混合细胞中验证基因敲入策略的可行性。并根据EGFP标记基因使用细胞流式分选技术在混合细胞基础上挑选基因定点敲入阳性的单克隆细胞株,通过PCR和Sanger测序验证单克隆细胞系构建是否成功。结果构建并验证了sgRNA切割效率,其中sgRNA3和sgRNA4剪切效率最高,为20.86%,成功构建了sgRNA双顺反子切割载体并验证了切割活性。成功构建外源DNA基因敲入载体,并在混合细胞中确定该基因敲入策略的可行性。通过细胞流式分选、PCR、Sanger测序成功筛选并验证出一株SP-10-shRNA在ZFX基因定点敲入的细胞系。最终利用CRISPR/Cas9技术成功构建SP-10-shRNA基因定点敲入猪X染色体阳性克隆细胞系,为实现猪后代性别的选择性偏移提供了重要参考。展开更多
Pulmonary nodules represent an early manifestation of lung cancer.However,pulmonary nodules only constitute a small portion of the overall image,posing challenges for physicians in image interpretation and potentially...Pulmonary nodules represent an early manifestation of lung cancer.However,pulmonary nodules only constitute a small portion of the overall image,posing challenges for physicians in image interpretation and potentially leading to false positives or missed detections.To solve these problems,the YOLOv8 network is enhanced by adding deformable convolution and atrous spatial pyramid pooling(ASPP),along with the integration of a coordinate attention(CA)mechanism.This allows the network to focus on small targets while expanding the receptive field without losing resolution.At the same time,context information on the target is gathered and feature expression is enhanced by attention modules in different directions.It effectively improves the positioning accuracy and achieves good results on the LUNA16 dataset.Compared with other detection algorithms,it improves the accuracy of pulmonary nodule detection to a certain extent.展开更多
文摘为了实现精子的内源性分离,本研究利用CRISPR/Cas9技术,通过基因双链断裂后的同源修复机制,将睾丸特异性启动子SP-10和干扰精子发育重要基因SPAG6、PPP1CC的shRNA偶联后定点整合进宁乡猪肾成纤维细胞X染色体的ZFX基因,构建并验证基因定点整合的细胞系。通过构建2个递送载体,根据人工多顺反子-tRNA-gRNA(PTG策略)基因串联表达系统原理,靶向ZFX基因设计多对sgRNA,验证sgRNA剪切效率,并构建sgRNA双顺反子切割载体。基于同源修复机制和同源介导末端连接(Homology Mediated End Joining,HMEJ)策略,构建含有新霉素抗性、EGFP标记基因等筛选标记的外源基因敲入载体,细胞电穿孔共转染后通过新霉素抗性筛选在混合细胞中验证基因敲入策略的可行性。并根据EGFP标记基因使用细胞流式分选技术在混合细胞基础上挑选基因定点敲入阳性的单克隆细胞株,通过PCR和Sanger测序验证单克隆细胞系构建是否成功。结果构建并验证了sgRNA切割效率,其中sgRNA3和sgRNA4剪切效率最高,为20.86%,成功构建了sgRNA双顺反子切割载体并验证了切割活性。成功构建外源DNA基因敲入载体,并在混合细胞中确定该基因敲入策略的可行性。通过细胞流式分选、PCR、Sanger测序成功筛选并验证出一株SP-10-shRNA在ZFX基因定点敲入的细胞系。最终利用CRISPR/Cas9技术成功构建SP-10-shRNA基因定点敲入猪X染色体阳性克隆细胞系,为实现猪后代性别的选择性偏移提供了重要参考。
文摘Pulmonary nodules represent an early manifestation of lung cancer.However,pulmonary nodules only constitute a small portion of the overall image,posing challenges for physicians in image interpretation and potentially leading to false positives or missed detections.To solve these problems,the YOLOv8 network is enhanced by adding deformable convolution and atrous spatial pyramid pooling(ASPP),along with the integration of a coordinate attention(CA)mechanism.This allows the network to focus on small targets while expanding the receptive field without losing resolution.At the same time,context information on the target is gathered and feature expression is enhanced by attention modules in different directions.It effectively improves the positioning accuracy and achieves good results on the LUNA16 dataset.Compared with other detection algorithms,it improves the accuracy of pulmonary nodule detection to a certain extent.
