Weiping YU

Animal Gene Editing Unit

Profile

Weiping YU

Weiping Yu received his BSc from Nankai University in 1983, MSc from Peking Union Medical College in 1986, and his Ph.D in Neurobiology from University College London in 1995. He then moved to IMCB for postdoctoral training first with Prof Catherine Pallen and later with Prof Byrappa Venkatesh. In 2003, he set up his own lab at the National Neuroscience Institute (NNI) where his research focuses on study of gene regulation mechanisms involved in the neurodegenerative diseases. In 2011, He moved to Biological Resource Centre (BRC), A*STAR, helping set up a service oriented Animal Gene Editing Laboratory (AGEL). In 2015, he took a joint position (while his primary employment is with BRC) and re-joined IMCB as a Group Leader in the Infrastructure, Technology & Translational Division.

Research

Animal Gene Editing Laboratory (AGEL)

Genetically modified animals are powerful tools for studying physiological function of genes in vivo in the whole animal context. The primary goal of AGEL is to provide services in creating genetically modified animal models and to conduct research on developing new gene editing technologies and novel animal models for human diseases. AGEL provides a wide spectrum of gene editing services in animals, including creation of transgenic rodents by pronuclear injection and gene knockin/knockout in mice via ES cell targeting. AGEL employs a number of gene editing methods including the recently developed TALEN and CRISPR/Cas9 nuclease-based techniques. We provide a comprehensive range of services that cover all of the steps involved in generating genetically modified animals including design of gene targeting strategy, synthesis and preparation of reagents and production of F1 heterozygotes etc.

Services Provided by AGEL

1) Gene targeting via ES cells

    • Design and construction of gene targeting vector
    • Generation of gene targeted ES cell lines
    • Creation of knockout/knockin chimeras by blastocyst injection
    • Production of F1 heterozygotes (germline transmission)

For floxed conditional knockout mice, we strongly advise researchers to search the IMPC database (https://www.mousephenotype.org/) to find out if the consortium has already generated targeted ES cell lines or knockout animals for the gene of interest.  Researchers may directly order targeted ES cells from the consortium, and AGEL can generate the knockout animals by blastocyst injection.  

2) Rodent transgenesis

    • Construction of tissue specific promoter driving transgene expression cassette
    • BAC engineering:  insertion of reporter gene, exon deletion or single/multiple nucleotide mutations
    • Stable transfection and screening for ES cells containing the genetically modified BAC
    • Creation of transgenic animals by pronuclear injection or blastocyst injection (BAC transgenesis)

Pronuclear injection of transgene construct is the traditional way to overexpress a protein coding gene or to express a reporter gene under a tissue-specific promoter in animals. However, the expression pattern of the transgene in animals generated by this method can be highly variable from line to line, and from generation to generation, due to random integration (positional and copy number) of the transgene. These issues occur less frequently (but are not completely eliminated) with BAC transgenesis.

3) Transgene expression at Rosa26 locus

    • Cre-mediated tissue/lineage-specific transgene expression at Rosa26
    • Tetracycline inducible transgene (protein-coding gene or shRNA) expression at Rosa26 with Cre-mediated tissue/lineage specificity

As a more precise alternative to pronuclear injection of expression constructs, we have developed a transgene expression platform that allows for a transgene cassette to be integrated as a single copy specifically at the Rosa26 locus. The expression of the transgene is under the control of a ubiquitous CAGGS promoter with a preceding floxed transcriptional stop signal inserted between the promoter and the transgene coding sequence. Tissue/lineage specificity of transgene expression is achieved by Cre-mediated deletion of the floxed “STOP”. Thus, by crossing with lines expressing Cre under the control of a range of tissue-specific promoters one can achieve different patterns of transgene expression with same animal model, ─ one model for multiple studies. Transgene expression can also be controlled temporally by tetracycline induction. The highly controllable (spatial and temporal) and predictable (single copy at predetermined genomic locus) nature of this system makes it ideal for studying the pathogenesis of gene mutations that may cause predisposition to human disease, because individual transgenic lines with different mutations can be directly compared for phenotypic differences. In addition to protein-coding genes, this system also supports shRNA expression under a well characterized miRNA expression cassette.  This facilitates acute knockdown of gene expression at any developmental stage (tetracycline-inducible) and in any tissue (with a specific Cre line of choice). Integration of individual transgenes at the docking site in ES cells is mediated by a highly efficient recombinase-mediated cassette change (RMCE) process, which allows for rapid generation of multiple transgenic lines simultaneously.


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4) TALEN & CRISPR/Cas9 mediated gene editing in mice and rats

    • Individual or large scale TALEN plasmid assembly and activity assay
    • TALEN or CRISPR/Cas9 mediated reading frame shift (indel) or single/multiple nucleotide mutagenesis in mice and rats

AGEL provides full support for the creation of genetically modified rodents using TALEN or CRISPR/Cas9 technology, including assembly and construction of specific TALEN plasmids, activity testing of TALENs and gRNA (see RESOURCES), reading frame shift mutagenesis in cells and animals (mice and rats), oligonucleotide-mediated single/multiple nucleotide mutagenesis in mice and rats etc. AGEL’s services are comprehensive, from design of the targeting strategy, synthesis and preparation of the reagents, microinjection of oocytes, to analysis of chimeric founders by deep sequencing and breeding of mutation segregated F1 heterozygotes.


(Click to view larger images)

5) Ad hoc services: In addition to the services listed above, AGEL provides advice and technical supports for custom’s projects relating to genetically modified animals and all enquiries are welcome. Please contact us AGEL-BRC@brc.a-star.edu.sg and we will get back to you as quickly as possible.

Staff

Department: Weiping YU

Name: Guisheng ZENG

Designation: Senior Research Fellow

Email: gszeng@imcb.a-star.edu.sg


Name: Yuan QIAO

Designation: Research Fellow

Email: yqiao@imcb.a-star.edu.sg


Name: Eve Wai Ling CHOW

Designation: Research Fellow

Email: wlechow@imcb.a-star.edu.sg


Name: Jiaxin GAO

Designation: Research Fellow

Email: gaojx@imcb.a-star.edu.sg


Name: Fong Yee CHAN

Designation: Laboratory Biologist A

Email: chanfy@imcb.a-star.edu.sg


Name: Xiao Li XU

Designation: Research Officer

Email: xiaoli@imcb.a-star.edu.sg


Name: Yue WANG

Designation: Research Director

Email: mcbwangy@imcb.a-star.edu.sg


Publications

Publications

Peer-Reviewed Research Articles

Wei Zhang, Selvaratnam Thevapriya, Paul J. Kim, Wei-Ping Yu, H. Shawn Je, Eng King Tan and Li Zeng (2014) 
Amyloid precursor protein regulates neurogenesis by antagonizing miR-574-5p in the developing cerebral cortex.
Nature Communications 5:3330

Yuen-Peng Tan, Shaobing Li, Xiao-Juan Jiang, Wailin Loh, Yik Khon Foo, Chay-Boon Loh, Qiurong Xu, Wai-Hong Yuen, Michael Jones, Jianlin Fu, Byrappa Venkatesh, Wei-Ping Yu  (2010)
Regulation of protocadherin gene expression by multiple neuron-Restrictive silencer elements scattered in the gene cluster.
Nucleic Acids Research 38:6985-4997

Xiao-Juan Jiang, Shaobing Li, Byrappa Venkatesh, Wei-Ping Yu  (2009)
Identification and comparative analysis of the protocadherin cluster in a reptile, the green anole lizard. PLoS ONE  4:e7614 

Wei-Ping Yu*, Viknesawari Rajasegaran, Kenneth Yew, Wai-lin Loh, Boon-Hui Tay, Chris T. Amemiya, Sydney Brenner*, Byrappa Venkatesh*  (2008)
Elephant shark sequence reveals unique insights into the evolutionary history of vertebrate genes: A comparative analysis of the protocadherin cluster.
Proc Natl Acad Sci U S A 105:3819-3824 (*Correspondence)

Paramasivam Kathirvel, Wei-Ping Yu, Byrappa Ventatesh, Chui-Chin Lim, Poh-San Lai and Woon-Chee Yee. (2008)
Fugu rubripes and human survival motor neuron genes: structural and functional similarities in comparative genome studies.
Gene  424:108-14

Wei-Ping Yu*, Kenneth Yew, Vikneswari Rajasegaran, Byrappa Venkatesh*. (2007)
Sequencing and comparative analysis of fugu protocadherin clusters reveal diversity of protocadherin genes among teleost.
BMC Evolutionary Biology 7:49 (*Correspondence)

Esther Wong, Wei-Ping Yu, Wai Ho Yap, Byrappa Venkatesh, Tuck Wah Soong.   (2006)
Comparative genomics of the human and Fugu voltage-gated calcium channel a1-subunit gene family reveals greater diversity in Fugu. 
Gene 366:117-127

Wei-Ping Yu, Jeanne M.M. Tan, Katherine C.M. Chew, Tania Oh, Prasanna Kolatkar, Byrappa Venkatesh, Ted M. Dawson, Kah Leong Lim (2005)
The 350-fold compacted Fugu parkin gene is structurally and functionally similar to human Parkin.
Gene 346:97-104

Wei-Ping Yu, Sydney Brenner and Byrappa Venkatesh. (2004)
Nested organization and evolution of vertebrate synapsin and Timp gene families.  
Journal of Neurochemistry 88 (supplement 1):37

Wei-Ping Yu, Sydney Brenner and Byrappa Venkatesh (2003)
Duplication, degeneration and complementation of the nested synapsin-Timp genes in Fugu rubripes
Trends in Genetics 19:180-183

Nigel P. Pringle, Wei-Ping Yu, Marisa Howell, Jennifer S. Colvin, David M. Ornitz and William D. Richardson (2003)
Fgfr3 expression by astrocytes and their precursors: evidence that astrocytes and oligodendrocytes originate in distinct neuroepithelial domains.
Development 130:93-102

Li Zeng, Si Xiaoning, Wei-Ping Yu, Hoa Thi Le, Kwok Peng Ng, Raymond M.H. Teng, Kenneth Ryan, Dennis Z.-M. Wang, Sathivel Ponniah and Catherine J. Pallen  (2003)
PTPa regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration
Journal of Cell Biology 160:137-146

Wei-Ping Yu, Catherine J. Pallen, Alice Tay, Frank R. Jirik, Sydney Brenner, Y.H. Tan, and Byrappa Venkatesh  (2001)
Conserved synteny between the Fugu and human PTEN locus and the evolutionary conservation of vertebrate PTEN function
Oncogene 20:5554-5561 

Lin Cui, Wei-Ping Yu, Catherine J. Pallen  (1998)
Insulin secretagogues activate the secretory granule receptor-like protein-tyrosine phosphatase IAR
Journal of Biological Chemistry  273:34784-34791

R.S. Schmidli, P.G. Colman, L. Cui, W.-P. Yu, K. Kewming, C. Jankulovski, L.C. Harrison, C.J. Pallen, and H.J. DeAzipurua  (1998)
Antibodies to the protein tyrosine phosphatases IA-2 and IAR are associated with progression to insulin-dependent diabetes (IDDM) in first-degree relatives at-risk for IDDM.
Autoimmunity   28:15-23 

Grant Morahan, Dexing Huang, Wei-Ping Yu, Lin Cui, Henry DeAizpurua and Catherine J. Pallen  (1998)
Localization of the genes encoding the type-1 diabetes autoantigens, protein-tyrosine phosphatases IA-2 and IAR.
Mammalian Genome   9:593-594

Andy Calver, Anita Hall, Wei-Ping Yu, Frank Walsh, John Heath, Christer Betsholtz and William D Richardson (1998)
Oligodendrocyte population dynamics and the role of PDGF in vivo.
Neuron  20:869-882 

W.D. Richardson, N.P. Pringle, W.-P. Yu, and A.C. Hall (1997)
Origins of spinal cord oligodendrocytes: possible developmental and evolutionary relationships with motor neurons.
Developmental Neuroscience  19:58-68  

Lin Cui*, Wei-Ping Yu*, Henry J. DeAizpuruz, Robert S. Schmidli and Catherine J. Pallen (1996)
Cloning and characterization of islet cell antigen-related proteiin-tyrosine phosphatase (PTP), a novel receptor-like PTP and autoantigen in Insulin-dependent diabetes.
Journal of Biological Chemistry  271:24817-24823 (*co-first authorship)

Nigel P. Pringle*, Wei-Ping Yu*, Sarah Guthrie, Henk Roelink, Andrew Lumsden, Alan C. Peterson, and William D. Richardson  (1996)
Determination of neuroepithelial cell fate: induction of the oligodendrocyte lineage by ventral midline cells and sonic hedgehog.
Developmental Biology  177:30-42 (*co-first authorship)

Wei-Ping Yu, Ellen J. Collarini, Nigel P. Pringle, and William D. Richardson. (1994)
Embryonic expression of myelin genes: evidence for a focal source of oligodendrocyte precursors in the ventricular zone of the neural tube.
Neuron  12:1353-1362

W.G. Li, Q.S Huang, B. Liu, Z.P. Ni, Q.N. Zhang, S.Y. Yu, Y.J. Zhu, C.Z. Huang, W.-P. Yu and C. Hou (1993)
Prevention of primary liver cancer by supplementation of selenium: the preliminary result of the first six years of study.
Chinese Journal of Cancer 02

S.Y. Yu, BL Mao, P. Xiao, W.-P. Yu, Y.L. Wang, C.Z. Huang, W.Q. Chen and X.Z. Xuan. (1990)
Intervention trial with selenium for the prevention of lung cancer among tin miners in Yunnan, China: A pilot study.
Biological Trace Element Research  24:105-108 

S.Y. Yu, W.G. Li, Y. J. Zhu, W.-P. Yu, C. Hou  (1989)
Chemoprevention trial of human hepatitis with selenium supplementation in China.
Biological Trace Element Research  20:15-22 



Review and Book Chapters

W.D. Richardson, N.P. Pringle, W.-P. Yu, E.J. Collarini and A. Hall. (1995).
Origins and early development of oligodendrocytes.
Glial Cell Development: Basic Principles and Clinical Relevance (Chapter Three). Bios Scientific Publishers

W.-P. Yu and S.Y. Yu.  (1986)
Oncomodulin and tumourgenesis. 
Progress in Biochemistry and Biophysics  13(6):12-1



Patents and Commercialization


Catherine J. Pallen, Lin Cui and Wei-Ping Yu.
IAR as a diagnostic reagent for insulin-dependent diabetes
(Patent: Pub No: WO/1997/022694, International Application No: PCT/CA1996/000867).

Wei-Ping Yu and Le-Ann Hwang.
Hybridoma cell lines for production of monoclonal antibodies against the common cytoplasmic domain of protocadherins alpha
(Licensed to Santa Cruz Biotechnology Inc. 2009, Product Cat#: sc-130555)

Wei-Ping Yu and Le-Ann Hwang.
Hybridoma cell lines for production of monoclonal antibodies against the common cytoplasmic domain of protocadherins gamma
(Licensed to Santa Cruz Biotechnology Inc. 2009, Product Cat#: sc-130556)

 

Resources

Resources

TALEN and CRISPR/Cas9 technologies

TALEN plasmid vectors: The vectors are constructed in a configuration similar to the widely used TALEN plasmid (Nat. Biotech. 29:143, 2011), but using our own proprietary TALEN sequences. The FokI nuclease domain is made of obligate heterodimers (Nature Method 8:74, 2011) and with sharkey mutations (J. Mol. Biol. 400:96, 2010). There are a total of three sets of vectors either with different promoters (CMV or CAGGS) or with the presence or absence of a different fluorescent tag (EGFP or mCherry) between the two obligate heterodimers. The fluorescent tags facilitate live cell imaging or cell sorting for isolating cells expressing both TALEN obligate heterodimers simultaneously. These vectors are compatible with the TALE tetramer library (see below) for assembly of TALEN in a single step (with 17 repeats) or two steps (with 18-21 repeats) of Golden Gate reaction. TALENs assembled with these vectors have successfully been used for mutating endogenous genes in cultured cells and in creating mutant rodents (both mice and rats) by pronuclear injection.

a) pCMV-TALEN (KKR-NG, KKR-NI, KKR-HD, KKR-NN, ELD-NG, ELD-NI, ELD-HD, ELD-NN)
b) pCAGGS-TALEN (NG, HD)
c) pCMV-TALEN-2A-tag (EGFP: KKR-NG, KKR-NI, KKR-HD, KKR-NN; mCherry:  ELD-NG, ELD-NI, ELD-HD,     ELD-NN)

TALE Tetramer Library: The library contains a total of 1024 tetramer clones. With the TALEN vectors described above the tetramer clones allow for extremely high fidelity of assembly of TALEN with 17-21 repeats by a single or two steps of Golden Gate reaction within 3 working days. Importantly, the library supports low cost, high-throughput TALEN assembly in any laboratory with basic molecular biology setup.  One person is able to generate 48 pairs of TALEN in a 96-well plate format in 3-4 working days.

Episomal reporters for TALEN activity assay: a simple double fluorescence episomal vector allows for cell-based (e.g. HEK293) TALEN activity testing. A derivative with drug resistance genes can be used as an episomal selection marker for TALEN-mediated endogenous gene mutagenesis in cultured cells.

Episomal reporters for gRNA assay: a simple fluorescence episomal vector allows for cell-based (e.g. HEK293) quick screening of a panel of candidate gRNAs.

Examples of Animal Models Currently Available

“Green mice”: with a single copy of the EGFP gene under a CAGGS promoter inserted at the Rosa26 locus. Heterozygotes (and homozygotes) exhibit a high level of EGFP expression in virtually all embryonic and adult tissues examined. These mice can be used as a source of donor tissues or cells for transplantation assays, allowing easy visualization and tracing of the transplanted material.

LRRK2 G2019S mice: with a glycine to serine mutation in the endogenous mouse LRRK2 gene at the position equivalent to human LRRK2 amino acid 2019, mimicking the human G2019S allele that results in predisposition to Parkinson’s disease. The model was created by TALEN-mediated single nucleotide mutagenesis and has two unique features over the commonly used LRRK2 models: 1) homozygotes only produce G2019S mutant but not wild type LRRK2; and 2) the mutant LRRK2 is expressed at the physiological level under control of the endogenous regulatory mechanisms. Thus, this model is a valuable tool for studying LRRK2 G2019S biology and pathology in vivo.