Analysis of DNA Methylation in Various Swine Tissues

Abstract

DNA methylation is known to play an important role in regulating gene expression during biological development and tissue differentiation in eukaryotes. In this study, we used the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) method to assess the extent and pattern of cytosine methylation in muscle, heart, liver, spleen, lung, kidney and stomach from the swine strain Laiwu, and we also examined specific methylation patterns in the seven tissues. In total, 96,371 fragments, each representing a recognition site cleaved by either or both EcoRI + HpaII and EcoRI + MspI, the HpaII and MspI are isoschizomeric enzymes, were amplified using 16 pairs of selective primers. A total of 50,094 sites were found to be methylated at cytosines in seven tissues. The incidence of DNA methylation was approximately 53.99% in muscle, 51.24% in the heart, 50.18% in the liver, 53.31% in the spleen, 51.97% in the lung, 51.15% in the kidney and 53.39% in the stomach, as revealed by the incidence of differential digestion. Additionally, differences in DNA methylation levels imply that such variations may be related to specific gene expression during tissue differentiation, growth and development. Three types of bands were generated in the F-MSAP profile, the total numbers of these three types of bands in the seven tissues were 46,277, 24,801 and 25,293, respectively.In addition, different methylation patterns were observed in seven tissues from pig, and almost all of the methylation patterns detected by F-MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrated that the F-MSAP technique can be adapted for use in large-scale DNA methylation detection in the pig genome.

Changes in methylation of genomic DNA from chicken immune organs in response to H5N1 influenza virus infection

“…Methylation-sensitive restriction endonucleases do not cut methylated regions; therefore, F-MSAP amplifies the digested gDNA into fragments of different sizes, following which the methylation level and patterns are analyzed. Compared to traditional MSAP, F-MSAP uses fluorescently labeled selective primers, is efficient, sensitive, time-efficient, safe, and automatic (Yang et al, 2011). So far, F-MSAP has been used in biological, medical, and agricultural sciences, among others.…”

“…Genomic DNA (gDNA) was extracted according to the method described by Yang et al (2011). The extracted gDNA was purified using the MicroElute DNA Clean-Up Kit (Omega, Hartford, CT, USA) according to the manufacturer protocols.…”

“…The digested products were ligated with 2.0 µL 10X buffer, 5 pmol EcoRI adapter, 50 pmol HpaII-MspI adapter, and 1.0 U T4 ligase at 37°C for 12 h. The adapters and primers for EcoRI and HpaII/MspI were previously described by Yang et al (2011), with some modifications (Table 1).…”

EGTA, a calcium chelator, affects cell cycle and increases DNA methylation in root tips of Triticum aestivum L.

“…However, the ability of each member of this isoschizomer pair to cut DNA depends on cytosine methylation conditions specific to each enzyme. If one or both cytosines are methylated in a single strand (hemi-methylation), but not both DNA strands, HpaII does not cut the strand, whereas MspI does not cut if the outer cytosine is methylated in one or both strands [28,[30][31][32][33]. Therefore, the amplification bands of the enzyme-digested products can show both the presence of DNA methylation and types of DNA methylation occurring [28,[30][31][32][33].…”

Morphological analysis and DNA methylation in Conyza bonariensis L. cronquist (Asteraceae) phenotypes

“…One of the methods used to detect methylation occurrence is the Methylation Sensitive Amplified Polymorphism (MSAP) technique, in which there is digestion of genomic DNA with restriction endonucleases that are sensitive to methylation, followed by amplification of restriction fragments (Yang et al 2011).…”