The total RNA was extracted from the PAMs (at least 1.0 × 106 cells) with TRIzol™ Reagent (Accurate Biotechnology, Changsha, China) [28 (link)], and the cDNA was prepared with Super Script II Reverse Transcriptase (Thermo Fisher Scientific). Five pairs of primers for cloning the porcine DNMT1 gene (shown in Table S1 ) were designed on the basis of the porcine DNMT1 cDNA sequence reported in the GenBank database (NM_001032355.1). All the PCR products were sequenced with gene-specific primers. All the images of agarose gel electrophoresis were captured with the Image Lab Software version 5.1 (Bio-Rad Laboratories, Hercules, CA, USA).
The amino acid sequences of the human (NP_001124295.1), mouse (NP_001300940.1), porcine (NP_001027526.1), and cloned porcine DNMT1 proteins were aligned with Clustal V and edited with Genedoc. A phylogenetic tree was constructed from the available DNMT1 proteins with the neighbor-joining method in MEGA version 6.06 [29 (link)].
The amino acid sequences of the human (NP_001124295.1), mouse (NP_001300940.1), porcine (NP_001027526.1), and cloned porcine DNMT1 proteins were aligned with Clustal V and edited with Genedoc. A phylogenetic tree was constructed from the available DNMT1 proteins with the neighbor-joining method in MEGA version 6.06 [29 (link)].
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