Generation of pluripotent embryonic stem cells useful for manufacturing transgenic rabbits – ORYCTOGENE-II

The rabbit is a relevant animal model of a wide range of human conditions such as cardiovascular diseases, hypertension and atherosclerosis, which are major causes of mortality in Western countries. Rabbit is gaining interest as a model for the study of other human diseases and indications like diabetes, ophthalmology, and CNS diseases. Rabbit also appears as a promising bioreactor for the preparation of biological drugs (recombinant proteins and vaccines) derived from serum or milk. One key issue in the generation of transgenic rabbits – and the subsequent creation of models of human diseases – is the availability of pluripotent stem cells that are needed to colonize the preimplantation embryo in order to generate germline chimaeras. Over the past 20 years this technology has been successfully developed in the mouse. The project proposed herein aims to transfer this technology to the rabbit. To reach this goal, ORYCTOGENE-II gathers two academic laboratories from INSERM (P. Savatier and B. Pain) with a long standing expertise in pluripotent stem cells, and one partner from the industrial sector (Transgenic Rabbit Models) with expertise in the production of transgenic rabbits by DNA microinjection.
The main objective of the ORYCTOGENE-II project is to produce rabbit ESCs and iPSCs that exhibit the features of naive pluripotent stem cells, and which are therefore capable of colonizing the preimplantation embryo and contributing to the development of all embryonic tissues including the germline. To achieve this goal, ORYCTOGENE-II will develop an innovative strategy, which combines the enforced expression of naive state-specific transcription factors, and the genetic selection of naive ESCs and iPSCs.
The cornerstone of the ORYCTOGENE-II project is the production of transgenic rabbits expressing fluorescent reporter/selectable genes under the control of naive state-specific transcriptional regulatory elements. The transgenic embryos and fetuses will be used to produce ESCs and iPSCs, respectively. In both cases, rare cells harboring a naive phenotype will be genetically selected. So as to increase the production of naive ESCs, embryos will be infected with lentiviral vectors that express regulatable forms of pluripotency-inducing transcription factors such as Klf2, Klf4, and STAT3. Given the difficulty to generate transgenic rabbits by DNA microinjection, such an experimental paradigm that combines genetic selection and forced expression of transcription factors in pre-implantation embryos has not been implemented so far.
Ultimately, we will produce and validate rabbit pluripotent stem cell lines, which can be used for gene targeting experiments and production of germline chimaeras. Beside applications in rabbit biotechnology, these cell lines will be of interest to the scientific community working in the stem cell field, due to the expected similarities between human and rabbit embryos. The industrial partner will be the exclusive operator of the intellectual property generated by the project. If successful, the ORYCTOGENE-II project will provide the proof-of-concept of novel animal models useful to the pharmaceutical industry and to the improvement of human health.