Figure 1-Total RNA isolated from mouse lung (lane 1), liver (lane 2), and heart (lane 3). A 100 bp ladder precedes lane 1.
Figure 2-DNased treated RNA
Figure 3-PCR products from cDNA created from lung(lanes 1-4), liver(lanes 5-8), and heart(lanes 9-12) using primers specific for Cx26(lanes 1,5,and 9), Cx37(lanes 2,6,and 10), Cx40(lanes 3,7,and 11), and Cx43(lanes 4,8,and 12). A 100 bp ladder precedes lane 1.
Figure 4-PCR test for presence of genomic DNA. Primers spanning a 1kb intron were used to test for the presence of genomic DNA in the lung (lane 1), liver (lane 2), and heart (lane 3) cDNA template. A 100 bp ladder precedes lane 1.
RNA extraction from the three tissues: heart, liver, and lung yielded good products (Figure 1). There did appear to be some DNA contamination, as evidenced by the presence of a band at the top of the gel. These bands are not easily identified in the reproduction of the picture, but were clearly visible under UV light and in the picture of the gel. The two bright bands correspond to the 28S and 18S ribosomal subunits. After Dnase treatment of the samples, the band of DNA contamination has been lost, with only a slight decrease in the intensity of the ribosomal subunit bands. (Figure 2).
Figure 3 represents the tissue specific expression of the 4 connexins in question (Cx-26, Cx-37, Cx-40, and Cx-43). Connexins 37, 40, and 43 have been amplified from the mRNA’s from each of the three tissues: heart, liver, and lung, while Cx-26 is present in the liver and the lung, but absent from the heart. Cx-26 from both the liver and the lung showed a weaker band than the other 3 connexins, and also a higher degree of non-specific binding not visible in this picture because the “Gene-Cleaned” PCR product was used to run on the agarose gel.
The bands present were amplified from cDNA synthesized from the mRNA present in the tissues, not genomic DNA(Figure 4). The PCR products using primers BFCx37IF and BFCx37IR which spanned intron 1 in Cx-37 were around 200 bp as would be expected from amplification of the portion of Cx-37 lacking an intron. If the intron was present, the band would have been 1.2kb, and the amplification would have been from genomic DNA, not cDNA.
Sequencing confirms the bands amplified correspond to the connexin gene targeted by the connexin specific primers(Data not shown). Each purified PCR product used as a template for sequencing showed high homology (>94% to the mouse connexin gene it was thought to be). The sequences recorded were compared to regions of the connexin gene that were significantly different from other connexin genes, thus confirming the presence of the varying connexin genes in the heart, liver, and lung.
The results of these experiments confirms the presence of Cx-37, Cx-40, and Cx-43 in all three tissues. As stated before, these three connexins are thought to be expressed in the endothelial lining of blood vessels, so there presence in all three tissues is not surprising. More intricate dissections would have to be performed to remove the blood vessels and study the expression in the tissue cells alone. The absence of Cx-26 in the heart also confirms previous studies which have shown it is not expressed in this tissue. It would be interesting to measure the relative amounts of these connexins using dilution’s of the template for PCR amplification and comparing the strength of the bands from each tissue. Expression of mutated connexins or their absence in a tissue have been correlated to many diseases including deafness when Cx-26 is misexpressed in the ear, as well as abnormal development of the heart when Cx-37, Cx-40, or Cx-43 are misexpressed (mutated or absent)[11-15]. It may turn out that particular combinations of connexons coming together to form gap junction are responsible for the movement of particular metabolites, so the identification of specific gap junctions, even if present in only small amounts appears to be important. If this is the case, we will need to determine what connexins are expressed in tissues and in what amounts. Once this has been determined, it may be possible to identify what molecules are permitted to pass through specific channels and this could be important in learning the exact causes for connexin-related disorders. It is the identification of connexin mRNA’s present in only small amounts which makes this RT-PCR technique so powerful in this field.
Sincere thanks go out to Sabrina Volpi, Ira Daly, and Rocco Coli for their patience and guidance throughout this project. Without their help, this project would not have been possible. Thanks also to Dr. Berish Y. Rubin for the use of the lab and support throughout the semester as well as for providing me with this opportunity to pursue this individual project. A special thanks to Iman for valuable discussions and expertise which were provided during my time in the lab.
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