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In vivo alteration of the keratin 17 gene in hair follicles by oligonucleotide‐directed gene targeting
Author(s) -
Fan W.,
Yoon K.
Publication year - 2003
Publication title -
experimental dermatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.108
H-Index - 96
eISSN - 1600-0625
pISSN - 0906-6705
DOI - 10.1111/j.0906-6705.2003.00099.x
Subject(s) - keratin , oligonucleotide , biology , microbiology and biotechnology , hair follicle , gene , genetics , endocrinology
Using intradermal injection of a chimeric RNA–DNA oligonucleotide (RDO) or a single‐stranded oligonucleotide (ssODN) into murine skin, we attempted to make a dominant mutation (R94P) in the conserved α‐helical domain of keratin 17 (K17), the same mutation found in pachyonychia congenichia type 2 (PC‐2) patients with phenotypes ranging from twisted hair to multiple pilosebaceous cysts. Both K17A‐RDO and K17‐ssODN contained a single base mismatch (C G C to C C C) to alter the normal K17 sequence to cause an amino acid substitution (R94P). The complexes consisting of oligonucleotides and cationic liposomes were injected to C57Bl/6 murine skin at 2 and 5 day after birth. Histological examination of skin biopsies at postnatal day 8 from several mice showed consistent twisted hair shafts or broken hair follicles at the sebaceous gland level and occasional rupture of the hair bulb or epidermal cyst‐like changes. In the injected area, the number of full anagen hair follicles decreased by 50%. Injection of the control oligonucleotide, identical to K17A‐RDO but containing no mismatch to the normal sequence, did not result in any detectable abnormality. The frequency of gene alteration was lower than 3%, according to the restriction fragment length polymorphism (RFLP) analysis of the genomic DNA isolated by dissection of hair follicles from slides. Although intradermal injection of K17A‐RDO or K17‐ssODN caused a dominant mutation in K17 affecting hair growth and morphology, these phenotypic changes were transient either due to the compensation of K17 by other keratins or the replacement of the mutated cells by normal surrounding cells during hair growth.