Hindiii and noti restriction enzymes

The PCR-amplified DNA fragment encoding mCherry was cloned into pRNA2-(A)₁₂₈. Briefly, EGFP coding sequence was replaced with mCherry by digestion of plasmid with HindIII and NotI restriction enzymes (New England Biolabs), and insertion of mCherry fragment in the vector. mRNA synthesis was performed using the new plasmid, pRNA2-(A)₁₂₈-mCherry, exactly as described for pRNA2-(A)128; see Methods section “mRNA synthesis by in vitro transcription”. Macrophages were transfected with 62.5 ng and 250 ng mRNA coding EGFP or mCherry by Lipofectamine MessengerMAX (Thermo scientific). Moreover, the homology value of the two proteins amino acid sequence was calculated by NCBI online blast tool.

Human RUNX1-MTG8 cDNA was PCR-amplified from pcDNA3.1-RUNX1-MTG8 [28 (link)], with primers introducing Xho I and Not I restriction sites (forward: 5′ – AGA TCT CGA GAT GCG TAT CCC CGT AGA TGC – 3′; reverse: 5′ – GAC AAG CGG CCG CCT AGC GAG GGG TTG TCT CTA – 3′), and inserted into compatible restriction sites of the pLNCX2 plasmid. Human CBFB-MYH11 cDNA was subcloned from the pGEM-CMVa-CBFB-MYH11 plasmid (kindly provided by Dr. Paul Liu, NIH, Bethesda, MD) by restriction digestion using Hind III and Not I restriction enzymes (New England BioLabs, Ipswich, MA) and inserted into compatible restriction sites of pLNCX2. The mouse KIT cDNA was subcloned from pCMV-Sport6-c-Kit (GE Dharmacon, Lafayette, CO) into compatible restriction sites of pLNCX2. The human KIT cDNA was PCR-amplified from pDNR-KIT (Dana-Farber/Harvard Cancer Center DNA Resource Core) with primers introducing Xho I and Not I restriction sites (forward: 5′ - AGA TCT CGA GAC CAT GAG AGG CGC TCG CGG CGC CT – 3′; reverse: 5′ – ACC TGC GGC CGC TCA GAC ATC GTC GTG CAC AAG C – 3′) and inserted into compatible restriction sites of pLNCX2. All plasmids were sequenced to confirm the absence of mutations.