Key Laboratory of Assisted Reproduction, Ministry of Education

6 ARTICLES PUBLISHED IN JoVE

Bioengineering

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

Rong Li^*1, Jinxin Miao^*1,2, Zhiqiang Fan¹, SeokHwan Song³, Il-keun Kong^3,4, Yaohe Wang^2,5, Zhongde Wang¹

¹Department of Animal, Dairy, and Veterinary Sciences, Utah State University, ²National Centre for International Research in Cell and Gene Therapy, Sino-British Research Centre, School of Basic Medical Sciences, Zhengzhou University, ³Department of Animal Science Division of Applied Life Science (BK21 Plus), Gyeongsang National University, ⁴Institute of Agriculture and Life Science, Gyeongsang National University, ⁵Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

Biochemistry

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Huijuan You^*1, Shimin Le^*2,3, Hu Chen^2,4, Linyan Qin¹, Jie Yan^2,4,5

¹School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, ²Mechanobiology Institute, National University of Singapore, ³Department of Physics, National University of Singapore, ⁴Research Institute for Biomimetics and Soft Matter, Fujian Provincial Key Lab for Soft Functional Materials Research, Department of Physics, Xiamen University, ⁵Center for Bioimaging Sciences, National University of Singapore

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Cancer Research

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

Xiaoxing Lv¹, Meiru Zhao¹, Yuting Yi¹, Lucheng Zhang¹, Yanfang Guan¹, Tao Liu¹, Ling Yang¹, Rongrong Chen¹, Jianhui Ma¹, Xin Yi¹

¹Geneplus-Beijing Institute

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

Bioengineering

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Jianwei Chen^*1,2,3, Ning Shao^*1,2,3, Jiaying Hu^*4, Rong Li⁴, Yuanshou Zhu^1,2,3, Dabing Zhang^4,5, Shujuan Guo^1,2,3, Junhou Hui⁶, Peng Liu⁶, Litao Yang⁴, Sheng-Ce Tao^1,2,3

¹Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, ²State Key Laboratory of Oncogenes and Related Genes, ³School of Biomedical Engineering, Shanghai Jiao Tong University, ⁴Collaborative Innovation Center for Biosafety of GMOs, National Center for the Molecular Characterization of Genetically Modified Organisms, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, ⁵Key Laboratory of Crop Marker-Assisted Breeding of Huaian Municipality, Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environmental Protection, ⁶Department of Biomedical Engineering, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Neuroscience

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Mengye Zhu¹, Daying Zhang¹, Sicong Peng², Nana Liu², Jing Wu², Haixia Kuang², Tao Liu^2,3

¹Department of Pain, First Affiliated Hospital of Nanchang University, ²Department of Pediatrics, First Affiliated Hospital of Nanchang University, ³Center for Laboratory Medicine, First Affiliated Hospital of Nanchang University

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

Medicine

OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery

Tao Liu^*1,2,3,4,5, Xueling Song^*1,2,3,4,5, Xiaoying Zheng^*1,2,3,4,5, Liying Yan^1,2,3,4,5, Caihong Ma^1,2,3,5, Rong Li^1,2,3,4,5, Jie Yan^1,2,3,4,5, Jie Qiao^1,2,3,4,5

¹Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, ²National Clinical Research Center for Obstetrics and Gynecology, ³Key Laboratory of Assisted Reproduction, Ministry of Education, ⁴Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, ⁵Research Units of Comprehensive Diagnosis and Treatment of Oocyte Maturation Arrest, Chinese Academy of Medical Sciences

In vitro maturation (IVM) before gynecological operation (OP-IVM) combines IVM following oocyte retrieval with routine gynecological surgery and serves as an extension of conventional IVM applications for fertility preservation.