RESUMO
OBJECTIVE@#To construct a eukaryotic expression vector of bromodomain-containing protein 7 (BRD7) with deletion of bromodomain (BRD7△brd) using the homologous recombination and reverse PCR amplification techniques.@*METHODS@#The linear DNA fragments of bromodomain-deleted mutation of BRD7 (pIRES2-EGFP- 3Flag/BRD7△brd) were amplified by one pair of reverse PCR primers using high-fidelity enzyme, and then these fragments were transformed into E.coli to obtain the eukaryotic expression vector expressing BRD7△brd protein based on homologous recombination and plasmid cyclization.@*RESULTS@#Bromodomain-deleted clones were identified by digestion with restrictive enzymes, and then the sequence and protein expression were further confirmed by sequencing and Western blot assays. The results suggest that pIRES2-EGFP-3Flag/BRD7△brd was successfully constructed.@*CONCLUSION@#We establish a simple and quick method to construct plasmids with pIRES2-EGFP- 3Flag/BRD7△brd using reverse PCR amplification and homologous recombination techniques. We also found that the concentration of template in PCR reaction system is one of the critical factors that affect the rate of homologous recombination. Of all, this improved technique could be widely used in the construction of gene mutations.
Assuntos
Humanos , Proteínas Cromossômicas não Histona , Genética , Escherichia coli , Genética , Recombinação Homóloga , Mutação , Plasmídeos , Reação em Cadeia da Polimerase , Métodos , Deleção de Sequência , Transfecção , MétodosRESUMO
<p><b>OBJECTIVE</b>To evaluate the feasibility, safety and short-term clinical outcomes of robot-assisted minimally invasive esophagectomy (RAMIE).</p><p><b>METHODS</b>Clinical data of 17 patients with esophageal cancer who received RAMIE between April 2016 and July 2016 were analyzed retrospectively.</p><p><b>RESULTS</b>The age of the patients ranged from 44 to 83. Six patients received neoadjuvant radiochemotherapy while 11 patients underwent surgery alone. All patients were performed by the robot-assisted thoraco-laparoscopic minimally invasive esophagectomy. In-hospital mortality was 0%. None was converted to open transthoracic or laparotomy approach. In the neoadjuvant radiochemotherapy group, 3 patients received pathological complete response while 2 patients were stage II(A and 1 patient was stage II(B. In the surgery alone group, 1 patient was stage I(A, 3 patients were stage II(A, 5 patients were stage II(B, 1 patient was stage III(A and 1 patient was stage III(B. The mean operation time was 195 minutes (range 145 to 305 minutes). The mean blood loss was 60 ml (range 30 to 200 ml). Mean lymph node harvest was 28 nodes. The rate of radical resection was 100%. Median ICU stay was 4.5 days (range 1 to 36 days), and median overall postoperative hospital stay was 15.2 days(range 9 to 45 days). Postoperative complication occurred in 4 (23.5%) patients, including 3 (17.6%) of lung lesion, 2 (11.8%) of hoarseness, 1 (5.9%) of chylothorax, while no anastomotic leakage and arrhythmia was observed.</p><p><b>CONCLUSION</b>RAMIE for esophageal cancer is feasible and safe with favorable early outcomes.</p>