Cloning of RYR2 Gene
Human RYR2 gene encoded a 15 kb ORF was cloned into a mammalian expression vector pcDNA5/TO
Recently we have cloned mouse and human RYR2 (mus musculus and homo sapiens ryanodine receptor 2, RYR2, NM_023868 and NM_001035 respectively) into the mmamalian expression vector pcDNA5/TO (invitrogen). RYR2, encoded 4966 amino acids, ~15kb bases, probably one of the longest genes in mammalian genomes. With intricacy details, both mouse and human RYR2 genes had been successfully cloned.
Start material: total RNA.
Insert: mouse and human RYR2
Problems and solutions:
Extreme long insert. reverse transcription is done with random primers and as many 15 overlapping fragments were amplified from cDNA pools. 15 overlapping fragments were assembled into two ~7.5 kp fragments and these two fragments were cloned into pSUPER respectively (a topoisomerase charged vector from Adexon). The inserts were sequenced and mutations were corrected. The two 7.5 kb fragment were further inserted into the expression vector pcDNA5/TO. The cloned gene was confirmed by DNA sequencing.
Instability of the clones. We found the 3' half 7.5kb of the gene was toxic to bacterial strains and instable during the culture. Most of the clones were deleted and some of them had proved host bacterial transposon insertions. In order to avoid the instability problems, we selected a panel of competent cells, including ABLE, SURE, XL10-Gold from Stratagene, CpyCutter from Epicentre and Stbl2 from Invitrogen. We determined that only Stbl2 performed well at low temperature.
Figure 1: Digestion pattern of pcDNA5/TO-hRYR2. DNA from mini-prep of 12 colonies were digested with HindIII. Only samples #3, 4, 8 are with right pattern. samples #1, 2, 5, 6, 7, 9 and 11 was severely deleted and sample #12 missed a small band of 0.8kb. M, DNA marker, GeneRuler, 1kb plus from Frementas.