Identification of differentially expressed genes in scarless wound healing utilizing polymerase chain reaction‐ suppression subtractive hybridization

Abstract

Wound healing in fetal skin is well known to proceed without scarring, whereas adult (postnatal) skin wound healing is accompanied by scar formation. To identify differentially expressed genes during fetal wound (FW) healing, we have used polymerase chain reaction-suppression subtractive hybridization. This technique allows for a comparative analysis across the entire transcriptome of FW vs. unwounded fetal control tissue, including even potentially novel sequences. Our subtractive hybridization protocol identified 15 clones that are overexpressed in healing FWs, and 20 clones that are underexpressed. These include genes with both known and unknown functions. We have confirmed the differential pattern of expression for four of these candidate genes: elongation factor 1 alpha, elongation initiation factor 4e, and two transcripts thus far known only as an expressed sequence tags. With this approach, we have also identified novel genes potentially involved in scarless wound healing.

Local Probiotic Therapy with Lactobacillus plantarum Mitigates Scar Formation in Rabbits after Burn Injury and Infection

“…Forward and reverse primers were purchased from Integrated DNA Technologies (Coralville, IA), and fluorocoupled Taqman probes were purchased from Applied Biosystems. The RT reaction (using reverse primer) and subsequent real-time PCR assays were performed as previously described [20][21][22][23]. Using the comparative critical cycle (Ct) method and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous control, the expression of the target genes was normalized, and the relative abundance was calculated.…”

Cloning and expression of rabbit CCT subunits eta and beta in healing cutaneous wounds

“…The wounding protocol for our New Zealand white rabbits, and RNA isolation/storage from fetal control (FC), fetal wound (FW), adult control (AC), and adult wound (AW) tissues was performed as previously described (Darden et al 2000;Kathju et al 2006;Satish et al 2008). In brief, pregnant New Zealand white rabbits at 20-21 days gestation were operated via midline laparotomy under general anesthesia.…”

Differential expression of chaperonin containing T-complex polypeptide (CCT) subunits during fetal and adult skin wound healing

“…We have previously investigated the molecular basis for scarless fetal wound healing in a rabbit model system using several expressomic techniques to identify differentially expressed candidate genes that may be critical to this unique physiology (Darden et al 2000;Kathju et al 2006). Differential display identified the eta subunit of the chaperonin-containing T-complex polypeptide (CCT) as specifically downregulated during fetal wound healing.…”