The overall aim of the EASE CRACK IT Challenge is to generate an approach that improves the implantation rates of early stage embryos when combined with extended in vitro culture and non-surgical embryo transfer techniques. Using surgical procedures, implantation rates for unmanipulated two-cell embryos are currently ~40%. However, micromanipulated embryos are less robust with ~25% currently giving rise to live offspring.
Genetically altered (GA) mice are used extensively to study the function and regulation of genes and their role in human development and disease. In 2015, approximately 50% of the animals used for scientific procedures in the UK were for the creation and breeding of genetically modified animals, the majority of which are mice (Home Office, 2015).
The generation of GA mouse models involves one of three techniques that use embryos at different stages of development:
- Pronuclear injections take advantage of one-cell embryos, where DNA or RNA is injected directly into the pronucleus of a fertilised egg. This technique allows scientists to use gene editing techniques like CRISPR/Cas9.
- In vitro fertilisation (IVF) is used to produce two cell embryos. This is a particularly useful technique because it allows scientists to archive or exchange mouse strains as sperm when it is appropriate to do so.
- Embryonic stem (ES) cell injection takes advantage of ES cell culture techniques that allow complex gene targeting events to be performed in vitro. Quality Control (QC) verified ES cells are injected into blastocysts (3.5 day old embryos).
Embryos from all three approaches are transferred into pseudopregnant recipients. The majority of embryo transfer procedures performed in mice use surgical techniques, with implantation into the oviduct or uterus via a laparotomy performed under general anaesthesia (Nagy et al., 2014). Although this technique is well understood and relatively high implantation rates can be achieved, it is an invasive procedure and can lead to discomfort and surgical complications. In recent years, efficient Non-Surgical Embryo Transfer (NSET) techniques have been developed (Steele et al., 2013; Cui et al., 2014) and specific transfer devices are now commercially available.
The NSET approaches pass a catheter through the cervix, allowing embryos to be deposited directly in the uterine cavity. No surgery is involved, offering significant welfare gains. In competent hands these devices work as well as surgical embryo transfer techniques. However, the NSET techniques can only be used to transfer late stage pre-implantation embryos (i.e. the blastocyst stage (E3.5)). This makes the technique suitable for transferring embryos after ES cell injection but it is not suitable for earlier embryonic stages including one and two-cell embryos generated from IVF and pronuclear injection programmes which still need to be transferred into the oviduct using surgical techniques.
Past experience has shown that extended culture of one and two-cell embryos generated from IVF programmes or following pronuclear injection severely compromises their implantation success. This means that these common techniques do not get used in conjunction with the current NSET systems. However, optimising embryo culture conditions is an area of intense research in both the mouse and clinical field. For example, a recent publication by Truong et al., has reported that adding antioxidants (e.g. acetyl-L-carnitine, N-acetyl-Lcysteine and α-lipoic acid) increases blastomere numbers in in vitro cultured embryos, as well as improving fetal development with increased crown-rump lengths and fetal weights. These data indicate that altering the redox potential of embryo culture systems may be advantageous when culturing embryos, prior to NSET. Other work has also shown that the addition of myo-inositol, a precursor of phosphoinositides, also significantly increases the number of blastomeres in embryos that had been produced by a microinjection technique called intracytoplasmic sperm injection (Colazingari et al., 2014).
The aim of this CRACK IT Challenge is to maximise the use of the NSET technique either by the development of a reliable system for culturing in vitro manipulated embryos through to the blastocyst stage or the development of an approach to non-invasively manipulate the uterine environment.
Developing a robust system that combines an improvement in in vitro embryo culture conditions with the use of contemporary NSET techniques would eliminate the need to use surgical embryo transfer when implanting one and two-cell embryos. This would have significant benefits to all of those involved in the development of GA mouse models in terms of improved animal welfare and reduced costs.
Removing the need for surgical embryo transfer would eliminate a significant number of invasive procedures. Based on a survey completed by the International Society for Transgenic Technologies between September 2008 and September 2009 (Fielder et al., 2010), it is estimated that in excess of 250,000 surgical embryos transfers are currently performed globally each year. The MRC’s Mary Lyon Centre performs in excess of 2,500 surgical embryo transfer procedures per year. With a viable alternative to surgical transfer of one and two-cell embryos, up to 90% of all invasive embryo transfer procedures could be substituted with a more refined technique.
The availability of techniques developed through this Challenge would also benefit other ongoing research efforts. For example, in recent years the GA mouse breeding community has made huge improvements in the way it exchanges (Kenyon et al., 2014) and stores mouse strains in repositories such as the European Mouse Mutant Archive (Guan et al., 2014). In both cases the preferred option is to use frozen sperm which needs to be recovered using IVF techniques to produce two-cell embryos.
The advent of new gene editing techniques like CRISPR/Cas9 also means that many transgenic laboratories have switched their activities away from ES cell/blastocyst injection and are now concentrating on one-cell injection techniques. This has the potential to increase the number of surgical embryo transfers performed unless NSET techniques can be optimised.
Single Phase Challenge winner
Project team led by
- Dr Virginia Pensabene, University of Leeds, £95,883.
Full Challenge information
The following Challenge Panel considered applications submitted to this Challenge:
|Professor Ian Kimber OBE (Chair)||University of Manchester|
|Dr Martin Fray (Sponsor)||MRC Harwell|
|Dr Sue Allen (Sponsor)||MRC Harwell|
|Dr Ben Davies||University of Oxford|
|Dr Michael Capaldi||Sunergos Innovations Ltd|
|Mr Ian Rosewell||The Francis Crick Institute|
|Dr Brendan Doe||Wellcome Trust Sanger Institute|