A HIT-trapping strategy for rapid generation of reversible and conditional alleles using a universal donor

Targeted mutagenesis in model organisms is key for gene functional annotation and biomedical research. Despite technological advances in gene editing by the CRISPR-Cas9 systems, rapid and efficient introduction of site-directed mutations remains a challenge in large animal models. Here, we developed a robust and flexible insertional mutagenesis strategy, homology-independent targeted trapping (HIT-trapping), which is generic and can efficiently target-trap an endogenous gene of interest independent of homology arm and embryonic stem cells. Further optimization and equipping the HIT-trap donor with a site-specific DNA inversion mechanism enabled one-step generation of reversible and conditional alleles in a single experiment. As a proof of concept, we successfully created mutant alleles for 21 disease-related genes in primary porcine fibroblasts with an average knock-in frequency of 53.2%, a great improvement over previous approaches. The versatile HIT-trapping strategy presented here is expected to simplify the targeted generation of mutant alleles and facilitate large-scale mutagenesis in large mammals such as pigs.

Following the completion of animal genome sequencing projects, rapid and efficient mutagenesis strategies are needed for analyzing gene function and for creating human disease models. Gene trapping is a high-throughput mutagenesis strategy whereby random vector insertion can be achieved across the mouse genome. A typical gene-trap vector contains a promoter-less reporter/selection gene flanked by an upstream splice acceptor (SA) and a downstream poly(A) signal. Upon insertion into an intron of a gene, the vector both inactivates the trapped gene and enables the gene-specific expression of a reporter gene (Gossler et al. 1989; Stanford et al. 2001). To date, gene-trapping approaches have been successfully applied toward large-scale mutagenesis in mouse embryonic stem cells (mESCs) and generation of gene knockout mice (Skarnes et al. 2004). The main drawback of random gene trapping is that gene-trap alleles are not specifically engineered to target genes of interest in advance. Therefore, methods to streamline the introduction of predesigned, site-specific modifications into the genome by homologous recombination would represent a significant technological advance. Previously, a hybrid approach combining gene targeting and gene trapping (targeted trapping) enabled mutation of expressed genes in mESCs with high efficiency, using a gene-trap construct flanked by homologous sequences of the target locus (Friedel et al. 2005). Also, homologous recombination is commonly used for creating conditional alleles, which is essential to avoid embryonic lethality and to study the stage- and tissue-specific functions of genes (Branda and Dymecki 2004). However, both standard gene trapping and targeted trapping are only suitable for genes expressed in embryonic stem (ES) cells. Furthermore, construction of targeting donor vectors with homology arms is labor intensive and costly, and the low efficiency of homologous recombination is also a rate-limiting step for gene targeting in mammalian genomes.Recently, by taking advantage of precise genomic double-strand breaks (DSBs) created by the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system (Ran et al. 2013; Doudna and Charpentier 2014; Hsu et al. 2014), homology-directed repair (HDR) efficiency was substantially enhanced (Porteus and Carroll 2005), and even donors with short homology arms (Orlando et al. 2010) or single-stranded DNA oligonucleotides (Chen et al. 2011; Quadros et al. 2017) were found to be compatible with site-specific integration. However, each targeting donor for HDR still needs to be customized with gene-specific homology sequences. Because of the lack of ES cells for certain animals such as pigs, sheep, and cattle, the genome must be edited either in a zygote embryo or in a somatic cell for somatic cell nuclear transfer (SCNT) (Reddy et al. 2020). It is still not feasible to achieve large-scale insertional mutagenesis including conditional knockouts in these important species with random gene trapping or HDR-based methods. Also, the problem of genetic mosaicism in embryo editing remains unresolved (Mehravar et al. 2019), prompting a need for technological advances to accelerate genetic modification in somatic cells.Alternatively, the generally more efficient nonhomologous end joining (NHEJ) pathway has been exploited for site-specific insertion of exogenous DNA by simultaneous cleavage of both donor plasmid and genome using programmable nucleases (Cristea et al. 2013; Maresca et al. 2013; Brown et al. 2016; Suzuki et al. 2016; Sawatsubashi et al. 2018). In contrast to HDR-based strategies, NHEJ-mediated insertions do not require gene-specific homology arms, enabling diverse sites to be targeted with a universal donor vector. Therefore, we speculated that a gene-trap cassette could be inserted into a specific locus easily through this mechanism in any cell type.Here, by combining NHEJ-mediated knock-in and gene trapping, we developed a strategy for targeted mutagenesis, especially in somatic cells with low HDR activity, referred to as HIT-trapping. By using a universal donor, this strategy allows us to (1) create null alleles, (2) produce a fluorescent reporter signal that could potentially allow cells with null alleles to be identified very quickly, and (3) produce reversible and conditional alleles that would be very helpful to have in most animal models but are often cumbersome to create.     

The Molecular Genetics of Marfan Syndrome

Marfan syndrome (MFS) is a complex connective tissue disease that is primarily characterized by cardiovascular, ocular and skeletal systems disorders. Despite its rarity, MFS severely impacts the quality of life of the patients. It has been shown that molecular genetic factors serve critical roles in the pathogenesis of MFS. FBN1 is associated with MFS and the other genes such as FBN2, transforming growth factor beta (TGF-β) receptors (TGFBR1 and TGFBR2), latent TGF-β-binding protein 2 (LTBP2) and SKI, amongst others also have their associated syndromes, however high overlap may exist between these syndromes and MFS. Abnormalities in the TGF-β signaling pathway also contribute to the development of aneurysms in patients with MFS, although the detailed molecular mechanism remains unclear. Mutant FBN1 protein may cause unstableness in elastic structures, thereby perturbing the TGF-β signaling pathway, which regulates several processes in cells. Additionally, DNA methylation of FBN1 and histone acetylation in an MFS mouse model demonstrated that epigenetic factors play a regulatory role in MFS. The purpose of the present review is to provide an up-to-date understanding of MFS-related genes and relevant assessment technologies, with the aim of laying a foundation for the early diagnosis, consultation and treatment of MFS.     

STAT3 couples with 14-3-3σ to regulate BCR signaling,B-cell differentiation,and IgE production

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BSL-2实验室开展SARS-CoV-2核酸检测过程中的生物安全管理

目的:总结生物安全二级（bio-safety level 2,BSL-2）实验室开展新型冠状病毒（severe acute respiratory syndrome coronavirus 2,SARS-CoV-2）核酸检测过程中的生物安全管理经验与效果。方法:检测样本来源于2020年2月21日到2020年11月5日送...     

Neutralizing antibodies for the prevention and treatment of COVID-19

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) initiates the infection process by binding to the viral cellular receptor angiotensin-converting enzyme 2 through the receptor-binding domain (RBD) in the S1 subunit of the viral spike (S) protein. This event is followed by virus–cell membrane fusion mediated by the S2 subunit, which allows virus entry into the host cell. Therefore, the SARS-CoV-2 S protein is a key therapeutic target, and prevention and treatment of coronavirus disease 2019 (COVID-19) have focused on the development of neutralizing monoclonal antibodies (nAbs) that target this protein. In this review, we summarize the nAbs targeting SARS-CoV-2 proteins that have been developed to date, with a focus on the N-terminal domain and RBD of the S protein. We also describe the roles that binding affinity, neutralizing activity, and protection provided by these nAbs play in the prevention and treatment of COVID-19 and discuss the potential to improve nAb efficiency against multiple SARS-CoV-2 variants. This review provides important information for the development of effective nAbs with broad-spectrum activity against current and future SARS-CoV-2 strains.     

几种实验动物心肌内肾上腺素能纤维分布

本文在荧光显微镜下观察大鼠、家兔、豚鼠和猫心肌不同部位肾上腺素能纤维 ,以探讨心肌内肾上腺素能纤维的分布。结果表明 ,心房肌、心室肌内均含有肾上腺素能纤维 ,只是密度不同     

椎动脉和第1颈神经穿经硬膜处的形态学特点及其与高血压颈枕痛的关系

目的　观察椎动脉和第1颈神经穿经硬膜处的形态特点及毗邻结构,探讨高血压合并枕颈部疼痛的发生机制。 方法　头颈部标本18具,解剖剥离法暴露,观察椎动脉穿经硬膜处部位及毗邻结构、椎动脉与第1颈神经（C1）的位置关系。 结果　椎动脉在寰枕外侧关节内后侧穿经硬膜,穿经处形成边缘光滑的圆孔,直径6.5~9.0 mm,硬膜与椎动脉外膜由纤维结缔组织连结。C1神经前后根自脊髓发出向外侧走行于椎动脉内侧并在其下方相伴共同穿经硬膜孔（100%）,其中与动脉壁相贴者66.7%（12例）;神经嵌入动脉壁者22.2%（4例）;隔有硬膜组织者11.1%（2例）。C1神经根出孔后走行于椎动脉与椎动脉沟之间。 结论　椎动脉穿经硬膜孔处位置固定,孔边缘致密,限制椎动脉扩张,利于颈椎活动时维持椎动脉供血,当全身血压波动时椎动脉管径不会产生明显变化,以维持后循环血液动力学稳定,但血压升高有可能将C1颈神经根卡压在硬膜边缘,椎动脉搏动刺激C1颈神经根导致椎枕肌痉挛,出现枕颈部疼痛。这可能是高血压合并枕颈部症状的形态学基础。     

轻度青少年特发性脊柱侧凸有限元模型构建及分析

目的　构建轻度青少年特发性脊柱侧凸患者的有限元模型,验证该模型的有效性,并进行有限元分析。 方法　建立1例轻度青少年特发性脊柱侧凸患者第6颈椎至第5腰椎的有限元模型,从几何形态及力学两方面对该模型进行有效性验证。分析该有限元模型在模拟前屈、后伸、左侧屈、右侧屈、左旋转、右旋转6种运动状态下,各椎体的应力变化。 结果　成功构建了轻度青少年特发性脊柱侧凸患者的有限元模型,模型总节点数为2561811个,总单元数为1547806个,并验证了该模型有效。模拟后伸、旋转活动时,畸形最明显处的椎体应力变化趋势与静态时相反。 结论　本实验所构建的轻度青少年特发性脊柱侧凸有限元模型有效,可进一步用于该疾病的相关研究。     

Recurrent chromosome changes in 62 primary gastric carcinomas detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been applied to detect recurrent chromosome alterations in 62 primary gastric carcinomas. Several nonrandom chromosomal changes, including gains of 8q (31 cases, 50%), 20q (29 cases, 47%) with a minimum gain region at 20q11. 2-q12, 13q (21 cases, 34%) with a minimum gain region at 13q22, and 3q (19 cases, 31%) were commonly observed. The regions most frequently lost included: 19p (23 cases, 37%), 17p (21 cases, 33%), and 1p (14 cases, 23%). High copy number gain (DNA sequence amplification) was detected in 6 cases. Amplification of 8q23-q24.2 and 20q11.2-q12 were observed in 3 cases. Gain of 20q and loss of 19p were confirmed by fluorescence in situ hybridization using corresponding bacterial artificial chromosomes (BAC) clones from those regions. The gain and loss of chromosomal regions identified in this study provide candidate regions involved in gastric tumorigenesis.     

TLR7 and TLR9 trigger distinct neuroinflammatory responses in the CNS

Toll-like receptors (TLRs) 7 and 9 recognize nucleic acid determinants from viruses and bacteria and elicit the production of type I interferons and proinflammatory cytokines. TLR7 and TLR9 are similar regarding localization and signal transduction mechanisms. However, stimulation of these receptors has differing effects in modulating viral pathogenesis and in direct toxicity in the central nervous system (CNS). In the present study, we examined the potential of the TLR7 agonist imiquimod and the TLR9 agonist cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) to induce neuroinflammation after intracerebroventricular inoculation. CpG-ODN induced a more robust inflammatory response than did imiquimod after inoculation into the CNS, with higher levels of several proinflammatory cytokines and chemokines. The increase in cytokines and chemokines correlated with breakdown of the blood-cerebrospinal fluid barrier and recruitment of peripheral cells to the CNS in CpG-ODN-inoculated mice. In contrast, TLR7 agonists induced a strong interferon β response in the CNS but only low levels of other cytokines. The difference in response to these agonists was not due to differences in distribution or longevity of the agonists but rather was correlated with cytokine production by choroid plexus cells. These results indicate that despite the high similarity of TLR7 and TLR9 in binding nucleic acids and inducing similar downstream signaling, the neuroinflammation response induced by these receptors differs dramatically due, at least in part, to activation of cells in the choroid plexus.