J Vet Sci.  2013 Jun;14(2):167-173. 10.4142/jvs.2013.14.2.167.

Identification of differentially expressed genes in gauze-exposed omentum of dogs using differential display RT-PCR

• Rahman MM
• Kim Y
• Byeon YE
• Ryu HH
• Kim WH
• Rayhan MU
• Kweon OK

• KMID: 1705518
• DOI: http://doi.org/10.4142/jvs.2013.14.2.167

Abstract

Molecular mechanisms governing peritonitis caused by the presence of aseptic gauze have remained unclear. To identify the genes involved, sterile gauze-exposed omentum was collected at 0, 6, 12, 24, and 48 h intervals, and analyzed by differential display RT(reverse transcription)-PCR. Among over 1,200 bands, 230 bands were found differentially expressed. These bands represented the fragment sizes of approximately 200 to 1,500 bp. The eight fragments were expressed differentially in the treatment group but not in the control. The sequences of two bands were similar to those of genes associated with the inflammatory process and a band was related to repair and regeneration process. Another one was related with spermatogonia and the rest four were unknown. Additionally, amplicons corresponding to the full-length sequences of two inflammatory gene fragments were synthesized by rapid amplification of cDNA end PCR. One showed 99% similarity to the major histocompatibility complex class II dog leukocyte antigen-DR beta chain and the other was canis familiaris proteasome beta type 3. Results of the present study suggested that sterile gauze induced the differential expression of genes in the omentum involved in inflammation and healing process.

Keyword

canine; DD RT-PCR; gauze; omentum; peritonitis

MeSH Terms

Animals
*Bandages
Base Sequence
DNA, Complementary/analysis
Dogs/*genetics/metabolism
Gene Expression Profiling/veterinary
Gene Expression Regulation
Histocompatibility Antigens Class II/*genetics/metabolism
Molecular Sequence Data
Omentum/*metabolism
Proteasome Endopeptidase Complex/*genetics/metabolism
RNA, Messenger/analysis
Reverse Transcriptase Polymerase Chain Reaction/veterinary
*Wound Healing
DNA, Complementary
Histocompatibility Antigens Class II
Proteasome Endopeptidase Complex
RNA, Messenger

Figure

• 

  Fig. 1 Representative images of the annealing control primer-based PCR products for identifying differentially expressed genes during inflammation induction in cases of canine peritonitis. RNA fingerprinting results for the control and treatment animals at 0, 6, 12, 24, and 48 h with GAPDH as the internal control. Amplified cDNA products correspond to gene transcripts in the omentum associated with the response to gauze implantation. Lane M: 100 bp size marker. The right arrow indicates bands that correspond to changes in cDNA expression. The cDNA band was excised from the gel for further cloning and sequencing.

• 

  Fig. 2 Bands generated by 5'end rapid amplification of cDNA end (RACE) for clones 250-48 and 400-46. (A) Clone 250-48. (B) Clone 400-46. PCR products were separated in a 2% agarose gel. Lane 2 in each gel contains the 5'RACE PCR products; Lane M: DNA ladder. The arrows indicate the 5'RACE products in the gels.

• 

  Fig. 3 The sequence of the 5'end for clones 250-48 and 400-46 synthesized from the omentum of dogs along with the full-length sequences are shown. The full-length sequence of clone 250-48 synthesized from the canine omentum is shown in (A) and that of clone 400-46 appears in (B). The underlined sequences correspond to annealing sites of the forward primers used for ACP-based full-length gene synthesis (5'RACE primer). The ATG start codon and TAA stop codon are shaded, and the boxed consensus sequence AATAA is the signal for polyadenylation.

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