Services and Priorities
The Gene Targeting and Transgenic Core's services provide for the production of transgenic mouse models using DNA microinjection and gene targeting in mouse ES cells. The Core's services are operated through the School of Medicine and Dentistry and are available to all faculty within the University of Rochester.
Transgenic mice with random integration of genes into the mouse genome are used to study normal as well as altered gene expression. To generate transgenic mice, DNA constructs are directly microinjected into the male pronuclei of mouse zygotes. The number of copies of integrated DNA can vary from one to several hundred, arranged primarily in tandem head to tail arrays. Transgenic mice are often generated to characterize the ability of a promoter to direct tissue-specific gene expression (e.g., a promoter can be attached to a reporter gene such as lacZ or GFP), and to examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals.
Gene Targeting in Embryonic Stem Cells
The conventional approach to generate genetically modified mice is through the use of ESCs. This technique utilizes the tissue culture system to modify and select ES cells that have received an exogenous DNA of interest through homologous recombination (HR). Once generated and characterized, the modified ESCs are injected into embryonic day 3.5 (E3.5) mouse embryos (blastocysts) to generate “chimeric” mice as donor embryos for ES cells and host blastocysts are obtained from different strains of mice carrying different eye or coat pigmentation. The mouse chimeras are bred to generate mice carrying modified genes. Gene targeted mice are often generated to examine the effects of gene deletion (knockout), mutation (knock-in mutagenesis), overexpressing and misexpressing endogenous or foreign genes (knock-in) at specific times and locations in the animals (conditional), and to characterize the ability of a promoter to direct tissue-specific gene expression (e.g., a promoter can be attached to a reporter gene such as lacZ or GFP).
Gene Editing UsingCRISPR/Cas9
The CRISPR/Cas9 system uses the Cas9 nuclease to facilitate RNA-guided site-specific DNA cleavage, resulting a double-strand break (DSB). This system uses two components, mammalian codon-optimized Cas9 protein and guide RNAs (gRNAs) to direct Cas9 protein tosequence-specifically cleave the targeted DNA. When Cas9/gRNAs designed to target a specific site in the mouse genome are directly injected into fertilized mouse oocytes, cleavage at the target site followed by imperfect repair through non-homologous end joining (NHEJ) will result in small deletions or insertions (indels) of one or more base pairs. If the cleavage site resides within the coding region of a gene, the indel will cause frame-shift mutations downstream of the site, resulting a knockout of the gene. If a repair template with homology to the target site is present during the repair process, the repair of the DSB can occur through homology directed repair (HDR). The process is error-free if the DNA template is used for repair is identical to the original DNA sequence at the DSB, or mutated DNA template can be used to introduce very specific mutations into the damaged DNA, generating a point mutation or knock-in. CRISPR/Cas9 approach has shorter timelines than ESC-based gene targeting method and is most suitable for generating deletions, point mutations, and insertions of small DNA fragments (such as loxP, FLAG, HA, V5, etc.).
The Core provides services on DNA vector construction, mouse embryo and sperm cryopreservation, re-derivation of mice to obtain Specific Pathogen Free (SPF) status, generation of congenic mice (backcrossing), colony management (breeding and genotyping), and providing mice of common mouse strains (C57BL/6J, ICR, Cre, and FLP; regular and pregnant females or litters).
A completed project application form complete with a valid UCAR approval number is required to initiate the Core's services. After receipt of application materials, the Core will review submitted materials and then the Scientific Advisory Committee will assign prioritization of approved applications.