Techniques

Overview of Technology

The TGE Core offers three different technical approaches for creating genetically engineered mice, these are currently also being used for making genetically engineered rats.

Making Transgenic Mice and Rats

Transgenic mice are being generated through the injection of defined DNA constructs that are commonly known as transgenes into the pronucleus of fertilized mouse or rat eggs. The injected eggs are transferred into the oviduct of a pseudo-pregnant surrogate female where they may progress through the gestational stages of development to term. A fraction of the pups born will have the transgene integrated into their genome, usually (but not always) as multiple tandemly arranged copies at a single site. These mice or rats are termed transgenic and should be capable of transmitting the transgene through the germline.

Transgenic mouse/rat technology has a broad spectrum of applications ranging from the analysis of gene function and regulation to modeling human diseases. A schematic overview of some common types of transgenic applications is presented below:

Tetracycline (Tet)-regulated and doxycycline (Dox) inducible transgenic systems permit tissue-specific transgene expression in a temporally controlled manner. Conventionally, this involves the generation of separate transgenic strains including a rtTA transactivator strain that expresses a reverse tetracycline-regulated activator under the transcriptional control of a tissue or cell type-specific promoter and a TRE transresponder strain that harbors the desired cDNA under the transcriptional control of a tetracycline response element (TRE). The Tet-On system is configured in such a way that the rtTA requires the presence of Dox to bind to the TRE, thus resulting in the expression of the cDNA (see image below).

As the generation of separate transactivator and transresponder strains and their required genetic crossing is cumbersome, time-consuming, and costly, the lab of the Core’s Director pioneered the use of a transgenic approach that involved an integrated Tet-On regulatory system. This system combines the rtTA and TRE components in a single transgene construct as exemplified by Transgelin (Tagln) promoter-controlled expression of rtTA combined with rtTA-regulated Hoxc11 expression upon Dox induction (see image below and Pruett et al. 2012: http://bio.biologists.org/content/early/2012/03/19/bio.2012039).

CRISPR/Cas-Mediated Genome Editing

CRISPR (clustered regularly interspaced short palindromic repeats) represents the molecular backbone of an adaptive bacterial immune system designed to neutralize invading phages and plasmids. Key components of this system have been adopted as powerful tools for genetic engineering and genome editing (the latter term reflects this system's capability to be used for rapid single nucleotide exchanges).

The basic principle of CRISPR/cas-mediated genome editing is as follows: A single guide RNA (sgRNA) directs a CRISPR-associated nuclease, most commonly Cas9, to a specific target DNA sequence via base pairing. The only sequence requirement is that the target sequence is located just 5 feet of a NGG protospacer adjacent motif (PAM). Cas9 will catalyze a double strand break (DSB) in the target DNA approximately three base pairs upstream of the PAM. This DSB will then be repaired by non-homologous end joint (NHEJ) repair, which causes small insertions or deletions (indels). Alternatively, in the presence of single stranded oligonucleotides (ssODNs) or dsDNA containing specific sequences to be inserted that are flanked by homology arms matching the sequences surrounding the break, the repair can be directed towards homologous recombination (HRD type of repair). Accordingly, the repair type can be instrumentalized for effecting different types of changes as desired; this is schematically summarized in the image below:

This core has each of the CRISPR/Cas9 genome editing approaches shown in the image in its service portfolio, for both mice and rats.

Assisted Reproductive Technology Services (ARTS)

The services offered include freezing of mouse sperm and embryos, storage in liquid nitrogen (LN2), in vitro fertilization (IVF) using either fresh or previously frozen sperm (sperm resuscitation), and embryo resuscitation (oviductal or uterine transfer of previously frozen embryos).