Jove-Journal of Visualized Experiments最新文献_第8页

Efficient Gene Knockdown in the Liver via Intrasplenic Injection of Adeno-Associated Virus Serotype 8 (AAV8)-Delivered Small Hairpin RNA. 通过脾内注射血清型8腺相关病毒（AAV8）递送的小发夹RNA在肝脏中高效敲除基因

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2024-11-01 DOI: 10.3791/67376

Xiaoqin Liu, Robert Y L Tsai

{"title":"Efficient Gene Knockdown in the Liver via Intrasplenic Injection of Adeno-Associated Virus Serotype 8 (AAV8)-Delivered Small Hairpin RNA.","authors":"Xiaoqin Liu, Robert Y L Tsai","doi":"10.3791/67376","DOIUrl":"10.3791/67376","url":null,"abstract":"The liver is a major organ that performs essential metabolic functions. Developing an efficient and safe method to knock down gene expression in the liver provides an important tool for determining gene function in liver pathophysiology. In this study, we describe a method for intrasplenic injection of adeno-associated virus serotype 8 (AAV8) engineered to express a small hairpin RNA (shRNA) against a target gene of interest, nucleostemin (NS). Intrasplenic injection of AAV8 expressing an NS-targeting shRNA (AAV8-shNS1) achieved the same knockdown efficiency of NS in the liver as did portal vein injection, compared to the injection of AAV8 expressing a scrambled sequence shRNA (AAV8-shScr). Furthermore, injection of the AAV8-shRNA virus triggered minimal inflammatory reactions in the liver parenchyma. Most importantly, this intrasplenic injection protocol was not technically demanding and caused minimal bleeding at the injection site, which is the leading cause of perioperative and postoperative mortality when performing portal vein injection. This study reports an improved and relatively safe method to achieve efficient gene knockdown in the liver.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649404","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Retinal and Choroidal Thickness Changes in Populations with Helicobacter pylori Infection by Swept-Source Optical Coherence Tomography. 通过扫源光学相干断层扫描观察幽门螺杆菌感染人群视网膜和脉络膜厚度的变化。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2024-11-01 DOI: 10.3791/65821

Lin Jiang, Pei Zhang, Luoziyi Wang, Qianlin Ji, Jing Jiang, Yu Zhang, Xin Che, Yiwen Qian, Qingjian Li, Zhiliang Wang

{"title":"Retinal and Choroidal Thickness Changes in Populations with Helicobacter pylori Infection by Swept-Source Optical Coherence Tomography.","authors":"Lin Jiang, Pei Zhang, Luoziyi Wang, Qianlin Ji, Jing Jiang, Yu Zhang, Xin Che, Yiwen Qian, Qingjian Li, Zhiliang Wang","doi":"10.3791/65821","DOIUrl":"10.3791/65821","url":null,"abstract":"Around half of the world's population is infected with Helicobacter pylori (H. pylori), which is closely related to several ocular diseases. The study aims to evaluate the retinal and choroidal thickness changes in subjects with H. pylori infection by swept-source optical coherence tomography (SS-OCT). The ophthalmic examination and 13C-urea breath test (13C-UBT) were performed on all subjects participating in the cross-sectional study. The participants were divided into H. pylori (+) and H. pylori (-) groups depending on the 13C-UBT results. This study covered 2574 right eyes from 2574 subjects with H. pylori infection and 2574 right eyes from 2574 age- and sex-matched individuals without H. pylori infection. Out of the nine sectors of the early treatment diabetic retinopathy study (ETDRS) grid, the maximum retinal thickness was in the inner superior sector, while the minimum was in the center sector. The maximum choroidal thickness was in the inner superior sector, while the minimum was in the outer nasal sector. The choroid of each area of the ETDRS subfield in the H. pylori (+) group was significantly thicker than that in the H. pylori (-) group, but retinal thickness did not show any difference between the two groups. Increased choroidal thickness may be an early indicator of H. pylori-associated retinal or choroidal diseases.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647854","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining. 用免疫染色法对小鼠视网膜血管周细胞进行三维可视化。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2024-11-01 DOI: 10.3791/67436

Shuang Zhang, Yuqing Wang, Zheng Wang, Yihan Liu, Jingjing Cui, Yuxin Su, Jingyan Han, Jia Wang, Wanzhu Bai

{"title":"3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining.","authors":"Shuang Zhang, Yuqing Wang, Zheng Wang, Yihan Liu, Jingjing Cui, Yuxin Su, Jingyan Han, Jia Wang, Wanzhu Bai","doi":"10.3791/67436","DOIUrl":"https://doi.org/10.3791/67436","url":null,"abstract":"Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor β to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649395","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A1 Pulley Reconstruction for Severe Trigger Finger. A1 滑轮重建术治疗严重的扳机指。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2024-11-01 DOI: 10.3791/66514

Minwei Yang, Xiaodi Zou, Yaozhao Dong, Weijie Zhou, Fangyu Yi, Sohaib Hasan Abdullah Ezzi, Vishnu Goutham Kota, Mohamed Hasan Abdulla Hasan Abdulla, Haiying Zhou, Alenikova Olga, Sahar Ahmed Abdalbary, Liang Jinhui, Hui Lu

{"title":"A1 Pulley Reconstruction for Severe Trigger Finger.","authors":"Minwei Yang, Xiaodi Zou, Yaozhao Dong, Weijie Zhou, Fangyu Yi, Sohaib Hasan Abdullah Ezzi, Vishnu Goutham Kota, Mohamed Hasan Abdulla Hasan Abdulla, Haiying Zhou, Alenikova Olga, Sahar Ahmed Abdalbary, Liang Jinhui, Hui Lu","doi":"10.3791/66514","DOIUrl":"10.3791/66514","url":null,"abstract":"The aim of this study was to evaluate the effectiveness of A1 pulley reconstruction in treating severe trigger fingers and to compare its outcomes with those of the traditional A1 pulley release technique. A total of 43 patients participated in the study, divided into two groups: 22 patients underwent A1 pulley reconstruction, while the remaining 21 patients received the standard A1 pulley release procedure. The outcomes were assessed using the Michigan Hand Outcomes Questionnaire (MHQ) 1 month post-surgery. The results demonstrated that patients who received the A1 pulley reconstruction reported significantly better outcomes. These included enhanced hand function and quality of life, reduced levels of pain, improved aesthetic appearances, and higher overall satisfaction when compared to the traditional release group. The findings suggest that A1 pulley reconstruction offers superior benefits over the standard release procedure for individuals suffering from severe trigger fingers, highlighting its potential as a more effective surgical intervention.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts. 用重组功能化蜘蛛丝制成的纳米纤维基底膜模拟物，置于定制的组织培养插件中。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2024-11-01 DOI: 10.3791/67116

Linnea Gustafsson, Savvini Gkouma, Alexander Jönsson, Martin Dufva, My Hedhammar

{"title":"Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts.","authors":"Linnea Gustafsson, Savvini Gkouma, Alexander Jönsson, Martin Dufva, My Hedhammar","doi":"10.3791/67116","DOIUrl":"https://doi.org/10.3791/67116","url":null,"abstract":"Replicating tissue barriers is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649408","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Application of DNA Barcoding to Identify Medicinal Plants. 应用 DNA 条形码鉴定药用植物。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2024-11-01 DOI: 10.3791/66925

Shuai Yang, Shilin Chen, Jing Mo, Junfei Liu, Qi Pan, Chi Song

{"title":"Application of DNA Barcoding to Identify Medicinal Plants.","authors":"Shuai Yang, Shilin Chen, Jing Mo, Junfei Liu, Qi Pan, Chi Song","doi":"10.3791/66925","DOIUrl":"10.3791/66925","url":null,"abstract":"Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration obstructs their development. DNA barcoding, a technique for species identification by standard DNA regions, facilitates prompt and accurate identification of traditional medicinal plants. The process of DNA barcoding entails six basic steps: 1) processing the medicinal plants, 2) extracting high-quality total DNA from the medicinal plants using centrifugal column method, 3) amplifying target DNA region internal transcribed spacer 2 (ITS2) with universal primers of plants and performing Sanger sequencing, 4) splicing and aligning sequence to obtain the target sequence, 5) matching the barcode sequence against the barcode library for identification, 6) aligning sequence, comparing intraspecific and interspecific variation, constructing phylogenetic neighbor-joining tree. As shown in the results, the universal primer can amplify the target region. Basic Local Alignment Search Tool (BLAST) demonstrates the percentage identified was 100%, and the neighbor-joining tree demonstrates that the splicing sequences were clustered with the A. sinensis OR879715.1 clade, and the clade support value is 100. This protocol provides a reference for applying DNA barcoding technology as an effective method to identify medicinal plants and adulterants.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649399","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

CIRCLE-Seq for Interrogation of Off-Target Gene Editing. CIRCLE-Seq 用于质询脱靶基因编辑。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2024-11-01 DOI: 10.3791/67069

Jeffrey Inen, Chann Makara Han, David M Farrel, Ganna Bilousova, Igor Kogut

{"title":"CIRCLE-Seq for Interrogation of Off-Target Gene Editing.","authors":"Jeffrey Inen, Chann Makara Han, David M Farrel, Ganna Bilousova, Igor Kogut","doi":"10.3791/67069","DOIUrl":"10.3791/67069","url":null,"abstract":"Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649400","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Establishment of A Mouse Model of Aqueous Deficiency Dry Eye. 建立缺水性干眼症小鼠模型

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2024-11-01 DOI: 10.3791/67317

Meng Zhang, Xiaoyu Tian, Yiming Wu, Liying Zhang, Lingli Zhang, Xueer Zheng, Shangkun Ou, Hao Gu

{"title":"Establishment of A Mouse Model of Aqueous Deficiency Dry Eye.","authors":"Meng Zhang, Xiaoyu Tian, Yiming Wu, Liying Zhang, Lingli Zhang, Xueer Zheng, Shangkun Ou, Hao Gu","doi":"10.3791/67317","DOIUrl":"10.3791/67317","url":null,"abstract":"Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its underlying mechanisms. Therefore, we developed a mouse model of dry eye disease by surgically removing both the extraorbital lacrimal glands (ELG) and intraorbital lacrimal glands (ILG) to investigate the ocular surface pathology in the context of aqueous deficiency dry eye. Two weeks post operation, the mice exhibited severe dry eye manifestations, including reduced tear secretion, corneal epithelial irregularities, positive fluorescein sodium staining, and neovascularization. Histological examination via hematoxylin and eosin staining revealed inflammatory cell infiltration and corneal epithelium dysplasia. Immunofluorescence staining and quantitative reverse-transcription polymerase chain reaction revealed decreased expression of the normal corneal epithelial biomarkers K12 and Pax6 and increased expression of Sprr1b in the corneal epithelium. These ocular manifestations indicated abnormal corneal epithelial differentiation. Furthermore, immunofluorescence staining of Ki67 revealed the increasing cell proliferation. In conclusion, the ELG plus ILG excision model proved suitable for studying changes in the ocular surface and elucidating the mechanisms underlying aqueous deficiency dry eye.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649407","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Establishing A Mouse Model for Respiratory Tract Extreme Low-temperature Air Inhalation. 建立呼吸道极端低温空气吸入小鼠模型

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2024-11-01 DOI: 10.3791/67254

Liang Yanchao, Xiang Huan, Liu Chunyang, Yuan Chao, Sun Jianda, Yang Guang

{"title":"Establishing A Mouse Model for Respiratory Tract Extreme Low-temperature Air Inhalation.","authors":"Liang Yanchao, Xiang Huan, Liu Chunyang, Yuan Chao, Sun Jianda, Yang Guang","doi":"10.3791/67254","DOIUrl":"https://doi.org/10.3791/67254","url":null,"abstract":"Currently, constructing a mouse model for cold environmental stimulation employs cold-heat plates and wearable cooling devices. These methods can partially fulfill the requirements for studying the responses and regulatory effects of mouse skin or neural circuits to cold stimulation. Numerous clinical studies have substantiated the correlation between exposure to low-temperature environments and the development of various diseases. Recently, there has been a growing emphasis on the continuous exchange of information between organs and tissues, providing a novel perspective on addressing longstanding issues within the human body. However, existing installations are unable to construct a model for mice inhaling cold air. Although placing mice in a cold environment seems attractive, it has considerable limitations. While mice inhale cold air, their skin is also being stimulated by the cold environment, making it unclear whether resulting pathological changes are due to lung stimulation through the interaction of distant organs or due to the skin receptors and neural signal transmission. This creates considerable confusion in related research. This scheme presents a new approach for constructing a mouse model for extreme cold air inhalation stimulation. This device allows mice to inhale extremely low-temperature gases while their bodies remain at a normal temperature. It maximizes the simulation of the stimulating effects of extreme ambient temperatures on mice and meets the research needs for studying the relationship between extreme environmental temperatures and related diseases.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649405","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions. 在无饲养条件下培养的人类多能干细胞中进行 CRISPR-Cas9 介导的基因缺失。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2024-11-01 DOI: 10.3791/67296

Muhammad Abid Sheikh, Farah Helena Afandi, Grazia Iannello, Barbara Corneo, Bright Starling Emerald, Suraiya Anjum Ansari

{"title":"CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions.","authors":"Muhammad Abid Sheikh, Farah Helena Afandi, Grazia Iannello, Barbara Corneo, Bright Starling Emerald, Suraiya Anjum Ansari","doi":"10.3791/67296","DOIUrl":"10.3791/67296","url":null,"abstract":"The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical application of this technique, particularly in pluripotent stem cells, presents certain challenges, such as being time- and labor-intensive and having low editing efficiency. Here, we describe the generation of a CRISPR-mediated gene knockout in a human embryonic stem cell (hESC) line stably expressing sgRNAs for the L2HGDH gene, using a highly efficient and stable lentiviral-mediated gene delivery system. The sgRNAs targeting exon 1 of the L2HGDH gene were chemically synthesized and cloned into the lentiCRISPR v2-puro vector, which combines the constitutive expression of sgRNAs with Cas9 in a highly efficient single-vector system to achieve higher lentiviral titers for hESC infection and stable selection using puromycin. Puromycin-selected cells were further expanded, and single-cell clones were obtained using the limited dilution method. The single clones were expanded, and several homozygous knockout clones for the L2HGDH gene were obtained, as confirmed by a 100% reduction in L2HGDH expression using Western blot analysis. Furthermore, using MSBSP-PCR, the CRISPR mutation site was mapped upstream of the PAM recognition sequence of Cas9 in the selected homozygous clones. Sanger sequencing was performed to analyze the exact insertions/deletions, and functional characterization of the clones was conducted. This method produced a significantly higher percentage of homozygous deletions compared to previously reported non-viral gene delivery methods. Although this report focuses on the L2HGDH gene, this robust and cost-effective approach can be used to create homozygous knockouts for other genes in pluripotent stem cells for gene function studies.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0