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Cyclooxygenase 1 mRNA expression is undetectable in Madin Darby Canine Kidney cells.

Pelletier G, Padhi BK - BMC Res Notes (2015)

Bottom Line: There are conflicting reports on the expression of COX-1 and COX-2 in MDCK cells and this lingering uncertainty about such important pharmacological targets may affect the interpretation of results obtained from this cell line.In order to definitively settle the issue of cyclooxygenase expression in MDCK cells, we designed PCR primers based on dog genomic sequences to probe COX-1 and COX-2 mRNA expression in MDCK cells and dog kidney.By improving the characterization of cyclooxygenase expression in MDCK cells, this study will contribute to a better understanding of the properties of this cell line and lead to improved experimental designs and data interpretations.

View Article: PubMed Central - PubMed

Affiliation: Hazard Identification Division, Environmental Health Science and Research Bureau, Health Canada, Environmental Health Centre, 50 Colombine Driveway, P.L. 0803B, Tunney's Pasture, Ottawa, Ontario, K1A 0L2, Canada. guillaume_pelletier@hc-sc.gc.ca.

ABSTRACT

Background: Madin Darby Canine Kidney (MDCK) cells form polarized epithelium in vitro and are routinely used in research fields ranging from protein trafficking to influenza. However, the canine origin of these cells also means that compared to man or mouse, genomic resources are more limited and performance of commercially available antibodies often untested. The synthesis of pro-inflammatory prostaglandins in the kidney is mediated by the constitutively expressed cyclooxygenase 1 and the inducible cyclooxygenase 2 (COX-1 and COX-2, respectively). There are conflicting reports on the expression of COX-1 and COX-2 in MDCK cells and this lingering uncertainty about such important pharmacological targets may affect the interpretation of results obtained from this cell line.

Results: In order to definitively settle the issue of cyclooxygenase expression in MDCK cells, we designed PCR primers based on dog genomic sequences to probe COX-1 and COX-2 mRNA expression in MDCK cells and dog kidney. We report that while COX-1 and COX-2 genes are both expressed in dog kidney, COX-1 expression is undetectable in MDCK cells.

Conclusions: By improving the characterization of cyclooxygenase expression in MDCK cells, this study will contribute to a better understanding of the properties of this cell line and lead to improved experimental designs and data interpretations.

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Related in: MedlinePlus

Expression of cyclooxygenases in MDCK cells and dog kidney. A) Schematic representation of the exonic structure of the PTGS1 transcript (coding for the COX-1 protein) in dog, mouse, rat and human. Exon structure and alignment were based on NCBI RefSeq NM_001003023, NM_08969, NM_017043 and NM_001271162, accessed on February 6, 2015. Exons are numbered and their lengths indicated along with the locations of the PCR primers used in this study. Exons are not all shown to scale. B) Representative gel showing the expression of COX-1 (PTGS1), COX-2 (PTGS2) and HPRT1 in MDCK cells (M) and dog kidney (K). Three regions of the PTGS1 gene, namely Exon 10-11 (E10-11), E8-9 and E4-5 were targeted for PCR amplification. The Cox1_E4-5 primers were also used to amplify genomic DNA from MDCK cells and dog kidney, generating a larger amplicon (380 bp) due to the inclusion of a 212 bp intron. A 50-bp DNA ladder (L) is shown on the left.
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Fig1: Expression of cyclooxygenases in MDCK cells and dog kidney. A) Schematic representation of the exonic structure of the PTGS1 transcript (coding for the COX-1 protein) in dog, mouse, rat and human. Exon structure and alignment were based on NCBI RefSeq NM_001003023, NM_08969, NM_017043 and NM_001271162, accessed on February 6, 2015. Exons are numbered and their lengths indicated along with the locations of the PCR primers used in this study. Exons are not all shown to scale. B) Representative gel showing the expression of COX-1 (PTGS1), COX-2 (PTGS2) and HPRT1 in MDCK cells (M) and dog kidney (K). Three regions of the PTGS1 gene, namely Exon 10-11 (E10-11), E8-9 and E4-5 were targeted for PCR amplification. The Cox1_E4-5 primers were also used to amplify genomic DNA from MDCK cells and dog kidney, generating a larger amplicon (380 bp) due to the inclusion of a 212 bp intron. A 50-bp DNA ladder (L) is shown on the left.

Mentions: The expression of COX-1 (PTGS1), COX-2 (PTGS2) and HPRT1 (hypoxanthine phosphoribosyltransferase 1) genes in MDCK cells and dog kidney were assessed under similar experimental conditions. As expected, primers for the housekeeping gene HPRT1 and COX-2 amplified their target sequences from both dog kidney and MDCK cells (Figure 1B). However, while the three primer sets for the COX-1 gene successfully amplified their target sequences from dog kidney mRNA, they failed to amplify mRNA from MDCK cells (Figure 1B). Further PCR investigation using Cox1_E4-5 primers (Table 2) to probe genomic DNAs amplified a 380 bp fragment (including 212 bp of COX-1 intronic sequences) from both dog kidney and MDCK cells (Figure 1B). Hence, although COX-1 mRNA is not detected in MDCK cells, COX-1 coding sequences are present in MDCK genome and the exact reason behind this lack of expression remains to be determined.Figure 1


Cyclooxygenase 1 mRNA expression is undetectable in Madin Darby Canine Kidney cells.

Pelletier G, Padhi BK - BMC Res Notes (2015)

Expression of cyclooxygenases in MDCK cells and dog kidney. A) Schematic representation of the exonic structure of the PTGS1 transcript (coding for the COX-1 protein) in dog, mouse, rat and human. Exon structure and alignment were based on NCBI RefSeq NM_001003023, NM_08969, NM_017043 and NM_001271162, accessed on February 6, 2015. Exons are numbered and their lengths indicated along with the locations of the PCR primers used in this study. Exons are not all shown to scale. B) Representative gel showing the expression of COX-1 (PTGS1), COX-2 (PTGS2) and HPRT1 in MDCK cells (M) and dog kidney (K). Three regions of the PTGS1 gene, namely Exon 10-11 (E10-11), E8-9 and E4-5 were targeted for PCR amplification. The Cox1_E4-5 primers were also used to amplify genomic DNA from MDCK cells and dog kidney, generating a larger amplicon (380 bp) due to the inclusion of a 212 bp intron. A 50-bp DNA ladder (L) is shown on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4375849&req=5

Fig1: Expression of cyclooxygenases in MDCK cells and dog kidney. A) Schematic representation of the exonic structure of the PTGS1 transcript (coding for the COX-1 protein) in dog, mouse, rat and human. Exon structure and alignment were based on NCBI RefSeq NM_001003023, NM_08969, NM_017043 and NM_001271162, accessed on February 6, 2015. Exons are numbered and their lengths indicated along with the locations of the PCR primers used in this study. Exons are not all shown to scale. B) Representative gel showing the expression of COX-1 (PTGS1), COX-2 (PTGS2) and HPRT1 in MDCK cells (M) and dog kidney (K). Three regions of the PTGS1 gene, namely Exon 10-11 (E10-11), E8-9 and E4-5 were targeted for PCR amplification. The Cox1_E4-5 primers were also used to amplify genomic DNA from MDCK cells and dog kidney, generating a larger amplicon (380 bp) due to the inclusion of a 212 bp intron. A 50-bp DNA ladder (L) is shown on the left.
Mentions: The expression of COX-1 (PTGS1), COX-2 (PTGS2) and HPRT1 (hypoxanthine phosphoribosyltransferase 1) genes in MDCK cells and dog kidney were assessed under similar experimental conditions. As expected, primers for the housekeeping gene HPRT1 and COX-2 amplified their target sequences from both dog kidney and MDCK cells (Figure 1B). However, while the three primer sets for the COX-1 gene successfully amplified their target sequences from dog kidney mRNA, they failed to amplify mRNA from MDCK cells (Figure 1B). Further PCR investigation using Cox1_E4-5 primers (Table 2) to probe genomic DNAs amplified a 380 bp fragment (including 212 bp of COX-1 intronic sequences) from both dog kidney and MDCK cells (Figure 1B). Hence, although COX-1 mRNA is not detected in MDCK cells, COX-1 coding sequences are present in MDCK genome and the exact reason behind this lack of expression remains to be determined.Figure 1

Bottom Line: There are conflicting reports on the expression of COX-1 and COX-2 in MDCK cells and this lingering uncertainty about such important pharmacological targets may affect the interpretation of results obtained from this cell line.In order to definitively settle the issue of cyclooxygenase expression in MDCK cells, we designed PCR primers based on dog genomic sequences to probe COX-1 and COX-2 mRNA expression in MDCK cells and dog kidney.By improving the characterization of cyclooxygenase expression in MDCK cells, this study will contribute to a better understanding of the properties of this cell line and lead to improved experimental designs and data interpretations.

View Article: PubMed Central - PubMed

Affiliation: Hazard Identification Division, Environmental Health Science and Research Bureau, Health Canada, Environmental Health Centre, 50 Colombine Driveway, P.L. 0803B, Tunney's Pasture, Ottawa, Ontario, K1A 0L2, Canada. guillaume_pelletier@hc-sc.gc.ca.

ABSTRACT

Background: Madin Darby Canine Kidney (MDCK) cells form polarized epithelium in vitro and are routinely used in research fields ranging from protein trafficking to influenza. However, the canine origin of these cells also means that compared to man or mouse, genomic resources are more limited and performance of commercially available antibodies often untested. The synthesis of pro-inflammatory prostaglandins in the kidney is mediated by the constitutively expressed cyclooxygenase 1 and the inducible cyclooxygenase 2 (COX-1 and COX-2, respectively). There are conflicting reports on the expression of COX-1 and COX-2 in MDCK cells and this lingering uncertainty about such important pharmacological targets may affect the interpretation of results obtained from this cell line.

Results: In order to definitively settle the issue of cyclooxygenase expression in MDCK cells, we designed PCR primers based on dog genomic sequences to probe COX-1 and COX-2 mRNA expression in MDCK cells and dog kidney. We report that while COX-1 and COX-2 genes are both expressed in dog kidney, COX-1 expression is undetectable in MDCK cells.

Conclusions: By improving the characterization of cyclooxygenase expression in MDCK cells, this study will contribute to a better understanding of the properties of this cell line and lead to improved experimental designs and data interpretations.

Show MeSH
Related in: MedlinePlus