Transgenic & Genome Editing Core

Overview of Services

The Transgenic and Genome Editing Core is a full-service facility supported by Augusta University, The Georgia Research Alliance, and user fees. The Core’s mission is to provide timely and cost-effective services in all phases of transgenic mouse production by pronuclear microinjection into zygotes, gene targeting in embryonic stem cells (ESCs), and gene editing using CRISPR/Cas9 approach to the research community at Augusta University as well as other academic institutions and for-profit companies. The Core also provides services on preserving your valuable lines by cryopreservation of sperms and embryos, and resurrecting mouse lines using in vitro fertilization (IVF) or embryo transfer. Additional services include rederivation to create pathogen-free mouse lines, developing molecular reagents for genetic engineering, full colony management services, and expert advice on mouse genetics and breeding schemes. The Core is fully equipped to carry out the all procedures of generating mouse models as well as related services.

PLEASE! Do not submit your request  before meeting with us!

ACKNOWLEDGEMENT:
All work performed by Augusta University Genome Editing Core should be acknowledged in scholarly publications, posters, and oral presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. When you use this resource, please be sure to include the following acknowledgement: 

We gratefully acknowledge the Genome Editing Core at Augusta University for generating the mouse models necessary to support this research.

Services

CRISPR/Cas9 Gene Editing

The CRISPR/Cas9 system uses the guide RNA (gRNA) in conjunction with the Cas9 endonuclease to create a double stranded break in the DNA at the targeted locus. The break will be repaired by non-homologous end-joining (NHEJ), which tends to add or delete base pairs resulting in a frame shift mutation (KO mouse). Alternatively, if a repair DNA is included in the injection mix, the DNA break can be repaired by homology directed repair (HDR) allowing for gene-targeted insertions (KI mouse).

CRISPR reagents are injected into fertilized one-cell-stage embryos, and the microinjected embryos are transferred into pseudopregnant mothers that develop, deliver and nurse mutant offspring. It takes about two months to generate a gene-targeted founder mouse with CRISPR technology. We have successfully used this technology to create the following mutations:

  • KO mutations
  • Single point mutation KI
  • Simultaneous Epitope (2-3X) KI
  • Insertion of epitope tags (<2 kb) to create tagged fusion protein
  • Gene Deletion (<1 kb in size)
  • Conditional KO: simultaneous insertion of LoxP sites of 5’ and 3’ of critical exon(s)

ES Cells-Based Gene Editing

The Transgenic and Genome Editing Core can develop mice with targeted mutations in the entire mouse or in specific tissues or cell types. It normally takes about a year to generate a gene-targeted mouse. Gene-targeted mouse ES cells are produced by electroporating a gene targeting construct into wild-type ES cells. The targeting construct typically contains selectable markers surrounded by two regions of homology to the targeted locus. In a fraction of those cells that take up the targeting construct, homologous recombination between the genome of the ES cell and the two regions of homology will result in the replacement of the targeted locus with the targeting construct.

Knock-out or knock-in mice are created by microinjecting these gene-targeted mouse ES cells into mouse blastocysts. The injected blastocysts are then transferred to pseudopregnant females and allowed to develop to term. The resulting pups are chimeras. The chimeras are then bred to establish the knockout mouse line.

Transgenic Project

In Transgenic and Genome Editing Core, transgenic mice are generated by pronuclear microinjection of foreign DNA fragments into one-cell-stage mouse embryos. On average, it takes about three to four months to generate a transgenic mouse strain.

The Core can assist investigators with the design of transgenic DNA constructs. Once a transgenic construct has been assembled, the investigator files a request for microinjection services. Core personnel then excise the transgene from the plasmid backbone and prepare it for microinjection. They also set up superovulation and mating to obtain one-cell-stage embryos for microinjection and prepare pseudopregnant surrogate mothers for embryo-transfer procedures.

The DNA construct is typically injected into fertilized one-cell-stage embryos. Microinjected embryos are transferred into surrogate mothers, which develop these embryos to term. Once pups are born from these eggs, tail biopsies are collected for preparation of genomic DNA. This DNA is screened by PCR analysis for the presence of transgenic DNA. Transgenic animals are then bred to establish a cohort of mice, which can be used to study the phenotypic consequences of transgene expression.

Mice and Other Services

The Transgenic and Genome Editing Core will distribute mice from the core's Shared Mouse Resource program. The program is created to help researchers share mouse models and generate novel mouse models through a cost-sharing mechanism. Genetically modified mouse models are commonly used in biomedical research. However, the generation, characterization, maintenance, and distribution of mouse models are not free but costly and time-consuming. In order to assist researchers with sharing the valuable mouse resource, the core acquires and creates novel mouse strains and maintain live colonies or cryopreserve these strains. These novel strains include research tool mice such Cre, Flp, and reporter knock-in stains, and conditional knockout mouse strains. Since the novel mouse strains are acquired or are created through a cost-sharing mechanism, individual users of these novel mouse strains will pay a small sharing fee. The sharing program operation is not subsidized by Augusta University or grants, user fees are essential to cover operating costs and to expand the repository by acquiring and creating new strains. In order to obtain the SMR mouse strain, the recipient scientist must agree and sign Uniform Biological Material Transfer Agreement.

The Core also provides services on preserving your valuable lines by cryopreservation of sperms and embryos, and resurrecting mouse lines using in vitro fertilization (IVF) or embryo transfer.

 

Leadership

Lin Gan, PhD
Professor
Director, Genome Editing Core
Augusta University
Department of Neuroscience and Regenerative Medicine
Phone: (706) 721-9635
E-mail: ligan@augusta.edu

Location and hours of operation

The Core consists of a wet lab for molecular biology, cell culture, and embryo microinjection in Room CA3056 of Department of Neurosciences and Regenerative Medicine, and an animal housing and procedure rooms in Suite CA1117.

Monday through Friday 8:00AM – 5:00PM. The core will follow the Augusta University calendar for holidays.

Links and Resources

https://www.augusta.edu/mcg/dnrm/transgenomecore.php

 


Service list


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