Biomedical papers - Ahead of Print

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. X:X | 10.5507/bp.2017.033

Cloning of intronic sequence within DsRed2 increased the number of cells expressing red fluorescent protein

Rishikaysh V. Pisala, Hana Hrebikovaa, Jana Chvatalovaa, Tomas Soukupa, Stanislav Filipb, Jaroslav Mokrya
a Department of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 500 03 Hradec Kralove, Czech Republic
b Department of Oncology and Radiotherapy, Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 500 03 Hradec Kralove, Czech Republic

Aim: Cloning of artificial intronic sequence within the open reading frame (ORF) of DsRed2 gene.

Method: Splice prediction software was used to analyze DsRed2 sequence to find an ideal site for cloning artificial intronic sequence. Intron was cloned within DsRed2 using cyclic ligation assembly. Flow cytometry was used to quantify the number of cells expressing red fluorescence.

Result: Sequencing data confirmed precise cloning of intron at the desired position using cyclic ligation assembly. Successful expression of red fluorescence after cloning of intron confirmed successful intron recognition and splicing by host cell line. Cloning of intron increased the number of cells expressing red fluorescent protein.

Conclusion: Cloning of intronic sequence within DsRed2 has helped to increase the number of cells expressing red fluorescence by approximately four percent.

Keywords: DsRed2, Cyclic ligation assembly (CLA), intron cloned DsRed2

Received: January 19, 2017; Accepted: July 11, 2017; Prepublished online: August 24, 2017


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