Abstract
Hairless mice have been widely applied in skin-related researches, while hairless pigs will be a useful model for skin-related study and other biomedical researches. Dickkopf-related protein 1 (DKK1) is inhibitor of Wnt signaling pathway. Transgenic mice expressing DKK1 transgene under control of a human keratin 14 (K14) promoter display hairless phenotype, which encouraged us to generate transgenic minipigs expressing pig DKK1 transgene under control of K14 promoter and finally achieve hairless minipigs. To generate transgenic cloned pigs, we constructed the lentiviral expression vector pERKDZG which contains two independent expression cassettes, the transcription of Tibet minipig DKK1 and EGFP genes are driven by K14 promoter, while mRFP is regulated under the control of Ef-1α promoter. Prior to generating the transgenic pig, the functionality of pERKDZG construct was verified by fluorescence assay and via checking pDKK1 expression. Subsequently, lentiviruses harboring ERKDZG transgene infected porcine embryonic fibroblasts (PEFs), followed by sorting RFP-positive PEFs by flow cytometry to obtain the purified PEFs carrying ERKDZG, designated DKK1-PEFs as donor cells used for somatic cell nuclear transfer (SCNT). Finally, we obtained 3 DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 and EGFP transgenes, but unfortunately, DKK1 transgenic cloned pigs don't display hairless phenotype as expected. Taken together, we achieve DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 transgene which provide a pig model for exploring DKK1 gene functions in pigs.
Highlights
At present, the minipigs are becoming the most commonly used large laboratory animals because the minipigs are small in body size, easy to operate, and share anatomical, physiological and biochemical similarities with humans
PERKDZG was constructed via multiple steps of cloning as described in Methods, and the map is shown in Figure 1A. pERKDZG contains two independent expression cassettes, the transcription of pig DKK1 (pDKK1) and EGFP genes are driven by a human keratin 14 (K14) promoter, while mRFP is regulated under the control of EF-1α promoter
To express pDKK1 and EGFP transgenes in the porcine skin, we chose the human K14 gene promoter (Figure 1A) because it has been demonstrated to be strongly active in the dividing cells of the epidermal basal layer, hair follicles and oral epithelia [18, 19]
Summary
The minipigs are becoming the most commonly used large laboratory animals because the minipigs are small in body size, easy to operate, and share anatomical, physiological and biochemical similarities with humans. Because of the remarkable similarities between porcine and human skin structure, pigs are often considered as an ideal model for skin-related studies, including skin grafting [1], cosmetic identification [2], ultraviolet radiation [3], skin cancer [4], burns [5, 6], frostbite [7], skin aging [8] and etc. Used small laboratory animals without hair include nude mice, SKH hairless mice and hairless guinea pigs, etc [9]. SKH hairless mice have been widely applied in skin-related researches (i.e., hair tonic effect, skin allergies, skin grafting treatment and ultraviolet radiation response, etc) [10]. Hairless animals can www.impactjournals.com/oncotarget eliminate the need for hair removal procedures before experiment and surgery, and avoid the skin damage caused thereby
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