A new genetic tool to improve immune‐compromised mouse models: Derivation and CRISPR/Cas9‐mediated targeting of NRG embryonic stem cell lines

• Published in: Genesis (New York, N.Y. : 2000) Vol. 56; no. 9; pp. e23238 - n/a
• Main Authors: Martin Gonzalez, Javier, Baudet, Aurélie, Abelechian, Sahar, Bonderup, Kasper, d'Altri, Teresa, Porse, Bo, Brakebusch, Cord, Juliusson, Gunnar, Cammenga, Jörg
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.09.2018; Wiley Subscription Services, Inc
• Subjects: Animal models; Animals; Basic Medicine; Breeding; Cell Line; Cell lines; Clinical Medicine; CRISPR; CRISPR-Cas Systems; CRISPR/Cas9; Derivation; Diabetes mellitus; Embryo cells; Embryogenesis; Embryonic Stem Cells; Embryos; ES cell derivation; Fertilization in Vitro; Fetuses; Gata-2; GATA2 Transcription Factor - genetics; Gene Targeting; Genomes; Hematologi; Hematology; hematopoiesis; Hematopoietic stem cells; Immunocompromised Host - genetics; immunodeficient mice; Implantation; In vitro fertilization; Klinisk medicin; Medical and Health Sciences; Medical Genetics; Medicin och hälsovetenskap; Medicinsk genetik; Medicinska och farmaceutiska grundvetenskaper; Mice; Mice, Inbred NOD - genetics; Mice, Inbred NOD - immunology; Models, Biological; Mutation; NRG; Nuclease; Phenotypes; Pluripotency; RAG1 protein; Rodents; Stem cell transplantation; Stem cells; Xenografts; Xenotransplantation; immunodeficient mice; Gata-2; NRG; hematopoiesis; CRISPR/Cas9; ES cell derivation; CRISPR; Cas9
• ISSN: 1526-954X; 1526-968X; 1526-968X
• DOI: 10.1002/dvg.23238

Summary Development of human hematopoietic stem cells and differentiation of embryonic stem (ES) cells/induced pluripotent stem (iPS) cells to hematopoietic stem cells are poorly understood. NOD (Non‐obese diabetic)‐derived mouse strains, such as NSG (NOD‐Scid‐il2Rg) or NRG (NOD‐Rag1‐il2Rg), are the best available models for studying the function of fetal and adult human hematopoietic cells as well as ES/iPS cell‐derived hematopoietic stem cells. Unfortunately, engraftment of human hematopoietic stem cells is very variable in these models. Introduction of additional permissive mutations into these complex genetic backgrounds of the NRG/NSG mice by natural breeding is a very demanding task in terms of time and resources. Specifically, since the genetic elements defining the NSG/NRG phenotypes have not yet been fully characterized, intense backcrossing is required to ensure transmission of the full phenotype. Here we describe the derivation of embryonic stem cell (ESC) lines from NRG pre‐implantation embryos generated by in vitro fertilization followed by the CRISPR/CAS9 targeting of the Gata‐2 locus. After injection into morula stage embryos, cells from three tested lines gave rise to chimeric adult mice showing high contribution of the ESCs (70%–100%), assessed by coat color. Moreover, these lines have been successfully targeted using Cas9/CRISPR technology, and the mutant cells have been shown to remain germ line competent. Therefore, these new NRG ESC lines combined with genome editing nucleases bring a powerful genetic tool that facilitates the generation of new NOD‐based mouse models with the aim to improve the existing xenograft models.