Transgenic expression of human cytoxic T-lymphocyte associated antigen4-Immunoglobulin (hCTLA4Ig) by porcine skin for xenogeneic skin grafting

Abstract

Porcine skin is frequently used as a substitute of human skin to cover large wounds in clinic practice of wound care. In our previous work, we found that transgenic expression of human cytoxicT-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong xenogeneic skin graft survival. In this work, using a transgene construct containing hCTLA4Ig coding sequence under the drive of human Keratine 14 (k14) promoter, hCTLA4Ig transgenic pigs were generated by somatic nuclear transfer. The derived transgenic pigs were healthy and exhibited no signs of susceptibility to infection. The hCTLA4Ig transgene was stably transmitted through germline over generations, and thereby a transgenic pig colony was established. In the derived transgenic pigs, hCTLA4Ig expression in skin was shown to be genetically stable over generations, and detected in heart, kidney and corneal as well as in skin. Transgenic hCTLA4Ig protein in pigs exhibited expected biological activity as it suppressed human lymphocyte proliferation in human mixed lymphocyte culture to extents comparable to those of commercially purchased purified hCTLA4Ig protein. In skin grafting from pigs to rats, transgenic porcine skin grafts exhibited remarkably prolonged survival compared to the wild-type skin grafts derived from the same pig strain (13.33 ± 3.64 vs. 6.25 ± 2.49 days, P < 0.01), further indicating that the transgenic hCTLA4Ig protein was biologically active and capable of extending porcine skin graft survival in xenogeneic wounds. The transgenic pigs generated in this work can be used as a reproducible resource to provide porcine skin grafts with extended survival for wound coverage, and also as donors to investigate the impacts of hCTLA4Ig on xenotransplantation of other organs (heart, kidney and corneal) due to the ectopic transgenic hCTLA4Ig expression.

Efficient generation of B2m-null pigs via injection of zygote with TALENs

“…To assess the functional consequences of the absence of MHC I complex in pig tissues on xenotransplantation, we performed skin grafting from pigs to mice as described previously3132. For improved graft survival, grafted skin pieces were covered with a dressing of several layers of gauze pieces and secured with a transparent film dressing (Fig.…”

“…Skin grafting was performed as previously described3132. Briefly, Split-thickness skin grafts were made from the full thickness skin pieces surgically collected from pigs after anesthesia.…”

Genetically engineered pigs as investigative and translational models in dermatology

“…They have permitted the creation of genetically modified pigs for studying diseases such as cystic fibrosis, Duchenne muscular dystrophy and diabetes. 4 Apart from a few examples, which include the overexpression of the hedgehog transcriptional activator Gli2 in porcine skin, 5 and the generation of pigs locally expressing immunomodulatory proteins for pig-to-human skin transplantation, 6 genetically modified pigs have seldom been employed for dermatological studies. This is particularly regrettable as experimental dermatology could greatly capitalize on the availability of tailored porcine models (Fig.…”

Xenotransplantation literature update, September–October 2014

“…Wang et al. have expressed the hCTLA4‐Ig transgene in pigs (under the keratin promoter) and measured skin graft survival in rats. As the authors hypothesized, the hCTLA4‐Ig transgene doubled the graft survival time to 13 days.…”

“…A comparison to the authors immune suppression protocol to those used clinically suggests that it may be critical to define the immune cell populations and activation states in relation to the time of rejection. Wang et al [13] have expressed the hCTLA4-Ig transgene in pigs (under the keratin promoter) and measured skin graft survival in rats.…”