Development of humanized mouse and rat models with full-thickness human skin and autologous immune cells.

The human skin is a significant barrier for protection against pathogen transmission. Rodent models used to investigate human-specific pathogens that target the skin are generated by introducing human skin grafts to immunocompromised rodent strains. Infection-induced immunopathogenesis has been separately studied in humanized rodent models developed with human lymphoid tissue and hematopoietic stem cell transplants. Successful co-engraftment of human skin, autologous lymphoid tissues, and autologous immune cells in a rodent model has not yet been achieved, though it could provide a means of studying the human immune response to infection in the human skin. Here, we introduce the human Skin and Immune System (hSIS)-humanized NOD-scid IL2Rγnull (NSG) mouse and Sprague-Dawley-Rag2tm2hera Il2rγtm1hera (SRG) rat models, co-engrafted with human full-thickness fetal skin, autologous fetal lymphoid tissues, and autologous fetal liver-derived hematopoietic stem cells. hSIS-humanized rodents demonstrate the development of human full-thickness skin, along with autologous lymphoid tissues, and autologous immune cells. These models also support human skin infection following intradermal inoculation with community-associated methicillin-resistant Staphylococcus aureus. The co-engraftment of these human skin and immune system components into a single humanized rodent model could provide a platform for studying human skin infections.

Gene Editing in Human Pluripotent Stem Cells: Choosing the Correct Path.

The recent emergence of targeted nucleases has opened up new opportunities for performing genetic modifications with human pluripotent stem cells (hPSCs). These modifications can range from the creation of a routine knock-out to the more challenging single point-mutation. For both the new and established user, deciding on the best approach for the specific modification of interest can be an arduous task, as new and improved technologies are rapidly and continuously being developed. The choices between the reagents and methodologies depends entirely on the end-goal of the experiments and the locus to be modified. Investigators need to decide on the best nuclease to use for each experiment from among Zinc-Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 that would result in the highest likelihood of success with the fewest pitfalls. Furthermore, there have been significant improvements over the first-generation nucleases, such as the development of the dimeric CRISPR RNA-guided Fok1 nucleases (RFNs, marketed as NextGEN™ CRISPR) that reduces the "off-target" mutation rate, providing further options for investigators. Should researchers need to perform a point mutation, then considerations must be made between using single-stranded oligo-deoxynucleotides (ssODN) as the donor for homology-directed repair or utilizing a selection cassette within a donor vector in combination with an excision-only piggyBac™ transposase to leave a seamless edit. In this review, we will provide a general overview of the current technologies, along with methodologies for generating point mutations, while considering both their pros and cons.