ABSTRACT
We developed a gene gun method for the transfer of human agouti signalling protein (ASP) cDNA to alter rat skin colour in vivo. Human ASP cDNA was cloned into a modified cytomegalovirus plasmid and delivered to the skin of Long-Evans rats by gene gun bombardment. Skin pigmentation, body weight and blood sugar of ASP cDNA-transfected rats were recorded against the control group, which were injected with plasmids encoding for green fluorescent protein. The treated skin showed lighter skin colour after 3 days of ASP gene transfection. This depigmentation effect was most prominent on day 14 and the skin gradually returned to its original pigmentation by day 28. Successful transfection of ASP gene in skin and hair follicles, as well as downregulation of melanocortin-1 receptor (MC1R) and tyrosinase expression upon treatment, was confirmed using immunohistochemistry and Western blot analysis. Body weight and blood sugar in the treated rats did not show statistically significant differences as compared to control groups. These observations demonstrate that gene transfer using the gene gun method can induce high cutaneous ASP production and facilitate a switch from dark to fair colour without systemic pleiotropic effects. Such a colour switch may be that ASP is acting in a paracrine fashion. In addition, this study verifies that ASP exerts its functions by acting as an independent ligand that downregulates the melanocyte MC1R and tyrosinase protein in an in vivo system. Our result offers new, interesting insights about the effect of ASP on pigmentation, providing a novel approach to study the molecular mechanisms underlying skin melanogenesis.
Subject(s)
Biolistics , DNA, Complementary/administration & dosage , Genetic Therapy/methods , Intercellular Signaling Peptides and Proteins/genetics , Skin Pigmentation/genetics , Agouti Signaling Protein , Animals , Gene Expression , Humans , Immunohistochemistry/methods , Intercellular Signaling Peptides and Proteins/analysis , Male , Monophenol Monooxygenase/analysis , Rats , Rats, Long-Evans , Receptor, Melanocortin, Type 1/analysis , Skin/chemistry , Transgenes , Tubulin/analysisABSTRACT
OBJECTIVE: To identify the site of fetal blood sampling (FBS) with lesser complications; and also analyses the reasons for targetting the intrahepatic vein (IHV) for FBS. METHODS: Fetal blood sampling (FBS) performed on 382 women over a period of 7 years at the National University Hospital, Singapore was analysed. FBS was performed from 13 weeks of gestational age onwards. In 76.4% (292 of 382) the intrahepatic part of the umbilical vein (IHV) was targetted; in 18.3% (70 of 382) percutaneous umbilical cord sampling (PUBS) was performed; in 5.2% (20 of 382) cardiocentesis was performed to obtain fetal blood. RESULTS: Multivariate analysis showed an increase in odds of fetal loss for umbilical cord and cardiocentesis groups compared with the IHV FBS group. It was statistically significant (p < 0.01) only in the cardiocentesis group for fetal loss at < 2 weeks of performing the procedure.
