Biomedical Chemistry: Research and /Content/2023/201/ 2023, 6(3), e0001

Improvement of the Exon Method for Rapid Synthesis of cDNA of the Rat Renalase Gene

V.I. Fedchenko*, A.A. Kaloshin. A.E. Medvedev

Institute of Biomedical Chemistry, Pogodinskaya Street, 10, Moscow 119121, Russia; *e-mail: valfed38@yandex.ru

Keywords:rat renalase gene; exon; exon assembly; PCR; gene expression

DOI:10.18097/BMCRM00201

The whole version of this paper is available in Russian.

We have improved our previously developed method of exon cloning of cDNA of eukaryotic genes to obtain the rat renalase gene cDNA. In contrast to the previously used step-by-step pairwise assembly of exons, in this work the procedure of full-length cDNA preparation was shortened due to simultaneous assembly of four neighboring exons at once (exons 1-4 and exons 6-9 of the rat renalase gene). The two obtained sequences (exons 1-4 and 6-9) were combined into a full-length cDNA of the rat renalase gene. The cDNA synthesized in this way was cloned into the prokaryotic vector pET-28a(+), which was then expressed in E. coli cells. The correctness of this approach was confirmed by sequencing resultant cDNA sequencing, which showed full (100%) identity with the nucleotide sequence available in the GenBank database (accession code: GenBankNM_001014167).
Figure 1. The scheme of exon organization of the rat genomic DNA of (Rattus norvegicus) (GenBank NC_051336.1) containing 272375 bp. Nine exons were identified on the genomic DNA by computational analysis (gene prediction method Gnomon). These exons include seven transcriptional variants of renalase mRNA: 1, 2, X2, X3, X4, X5, X6.
Figure 2. The scheme of rat renalase gene cloning into the plasmid vector pET-28a(+) to obtain the vector pET-RenRatV2.
Figure 3. Electrophoresis in 2% agarose gel of DNA amplicons of the renalase exons obtained by PCR amplification with the total rat genomic DNA. Track: L - DNA marker: 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp The exon number corresponds to the track number in the gel. Estimated DNA sizes of individual amplicons, tracks: 1) - 135 bp, 2) - 128 bp, 3) - 159 bp, 4) - 182 bp, 6) - 199 bp., 7) – 196 bp, 8-9) – 92 bp.
Figure 4. 2% agarose gel electrophoresis of assembled DNA amplicon products of the renalase exons. Tracks: 1) – assembly of exons (ex-1, ex-2, ex-3 and ex-4) with an estimated amplicon DNA size of 537 bp; 2) – assembly of exons (ex-6, ex-7 and ex-8+ex-9) with an estimated amplicon DNA size of 459 bp; 3) – assembly of all exons (amplicon of exons 1-4 with amplicon of exons 6-9) with an estimated DNA amplicon size of 962 bp; L) - DNA marker: 50 bp, 100 bp, 200 bp. , 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp.
Figure 5. 2% agarose gel electrophoresis of assembled DNA amplicon products of the renalase exons. Tracks: 1) – assembly of exons (ex-1, ex-2, ex-3 and ex-4) with an estimated amplicon DNA size of 537 bp; 2) – assembly of exons (ex-6, ex-7 and ex-8+ex-9) with an estimated amplicon DNA size of 459 bp; 3) – assembly of all exons (amplicon of exons 1-4 with amplicon of exons 6-9) with an estimated DNA amplicon size of 962 bp; L) - DNA marker: 50 bp, 100 bp, 200 bp. , 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp.

CLOSE
Table 1. Primers used for PCR amplification to obtain exons (ex-1, ex-2, ex-3, ex-4, ex-6, ex-7, and ex-8+ex-9) of the mRNA transcription variant-2 of the rat renalase gene (Rattus norvegicus).

FUNDING

The work was performed within the framework of the Program for Basic Research in the Russian Federation for a long-term period (2021-2030) (№ 122030100170-5)

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