The Transgenic and Gene Targeting Facility (TGTF) is located at Clinical Research Centre (CRC), MD11, in NUS. The facility is equipped with Leica AM6000 micromanipulator, Eppendorf FemtoJet micro-injector, micro-pipette fabrication devices and tissue culture system.
The purpose of the TGTF is to provide technologically advanced and efficient mouse/rat services to the biomedical research community in Singapore. To modify mouse/rat genomes, we perform CRISPR/Cas9 (with or without DNA) injection for gene targeting or traditional DNA microinjection for transgenic services. We perform gene targeting in ES cells followed by blastocyst injection to generate chimeric mice. TGTF also offers assisted reproductive services, including embryo and sperm cryopreservation and storage, in vitro fertilization and rederivation and rescue of mouse/rat lines.
Dr. Yvonne Tay
CSI Singapore
yvonnetay[at]nus.edu.sg
Yu Shuizhou
Senior Laboratory Executive
csiyus[at]nus.edu.sg
TGTF – MD6, 13S, Bench-22
Shi Jizhong Edward
Senior Laboratory Executive
csisj[at]nus.edu.sg
TGTF – MD6, 13S, Bench-22
TGTF provides conventional transgenic mouse service by injecting transgenic DNA fragments or BAC DNA into mouse pronuclei. The injected DNA will be randomly integrated into the mouse genome; therefore, transgenic mice will be confirmed by genotyping only. We do not guarantee gene expression from the injected DNA fragment.
Direct and precise modification of the genomes of mouse and rat embryos. CRISPR/Cas9 technology has revolutionized rodent model production and is evolving rapidly. It has the following key advantages:
Success rate and projected timeline:
TGTF will harvest blastocysts from uteri of up to 5 pregnant E5 females. Blastocysts will be rinsed with M2 medium and placed onto mitomycin-C treated SNL feeder cell layers in embryo growth media with LIF. The blastocysts will be grown ex vivo for 4-5 days, when inner cell mass (ICM) will have expanded. ICM cells will be disaggregated and seeded in 96-well plate. TGTF will freeze 4 ES cell clones for customers and offer 106 ES cells of each clone for karyotype analysis by the PI’s lab.
Gene targeting in ES cells utilizes homologous recombination to replace targeted regions of endogenous DNA with engineered DNA in ES cell lines to generate knock-out, knock-in, point mutant or conditional alleles.
Optional add-on service requests:
Chimera mice will be generated by blastocyst injection of gene targeted ES cells . Up to 200 blastocysts will be injected with 2 positive ES cell clones for each project. For gene targeted ES cells generated at TGTF, we will inject an additional clone, into up to 100 blastocysts if no chimera mice were obtained from injection of the first 2 clones.
We default freeze 8-10 straws of sperm from one donor of each line. Two male mice at the age between 3-8 months are needed. Males with previous mating history are preferred.
Embryo Cryopreservation: 180-220 embryos at 2-cell stage will be cryopreserved in 4-5 cryovials for the PI’s lab. The PI’s lab has the following options:
Sperm and embryo cryobank storage service provides the PI’s labs a convenient, safe, short/long term storage of their mouse lines at a very low cost.
Rederivation involves the revival of mouse strains from cryopreserved sperm or embryos, the rescue of mouse lines from sterile old male mice, or the clean up of dirty mouse strains. The successful rate at TGTF is over 99%. The PI’s lab has the following options:
We will transfer embryos (frozen or fresh) into pseudopregnant female’s oviducts or uterus for gestation. Animals resulting will be transferred to the PI’s lab for genotyping and breeding.
TGTF will use fresh or previously frozen sperm to fertilize oocytes obtained from up to 10 superovulated females either from TGTF or from the PI’s lab. Embryos surviving to the two-cell stage can be transferred to the PI’s lab or implanted into pseudopregnant females for gestation or frozen for later implantation.
1. Aled O’Neill, Desmond Chin, Darren Tan, A’Qilah Banu Bte Abdul Majeed, Ayako Nakamura-Ishizu, and Toshio Suda. Thrombopoietin Maintains Cell Numbers of Hematopoietic Stem and Progenitor Cells With Megakaryopoietic Potential. Haematologica. 2020 Jun 11:haematol.2019.241406. doi: 10.3324/haematol.2019.241406.
2. Pavithra Shyamsunder, Mahalakshmi Shanmugasundaram, Anand Mayakonda, Pushkar Dakle, Weoi Woon Teoh, Lin Han, Deepika Kanojia, Mei Chee Lim, Melissa Fullwood, Omer An, Henry Yang, Jizhong Shi, Mohammad Zakir Hossain, Vikas Madan, H Phillip Koeffler. Identification of a Novel Enhancer of CEBPE Essential for Granulocytic Differentiation. Blood. 2019 Jun 6;133(23):2507-2517. doi: 10.1182/blood.2018886077.
3. Junichi Matsuo, Shunichi Kimura, Akihiro Yamamura, Cai Ping Koh, Md Zakir Hossain, Dede Liana Heng, Kazuyoshi Kohu, Dominic Chih-Cheng Voon, Hiroshi Hiai, Michiaki Unno, Jimmy Bok Yan So, Feng Zhu, Supriya Srivastava, Ming Teh, Khay Guan Yeoh, Motomi Osato, Yoshiaki Ito. Identification of Stem Cells in the Epithelium of the Stomach Corpus and Antrum of Mice. Gastroenterology. 2017 Jan;152(1):218-231.e14. doi: 10.1053/j.gastro.2016.09.018.
4. Desmond Wai Loon Chin, Naoko Watanabe-Okochi, Chelsia Qiuxia Wang, Vinay Tergaonkar, Motomi Osato. Mouse models for core binding factor leukemia. Leukemia. 2015 Oct;29(10):1970-80. doi: 10.1038/leu.2015.181.
Dry shipping is a convenient method for transportation or distribution of cryopreserved sperm or embryo internationally but sperm or embryo handling must be extremely careful for protection of the precious mouse lines in the straws or cryovials.
The PI’s lab will be provided with genotyping results in PPT slides with corresponding interpretations.
Please contact us (Edward Shi: csisj@nus.edu.sg) for further information and price list.
Cancer Science Institute of Singapore
Centre for Translational Medicine
14 Medical Drive, #12-01,
Singapore 117599