A highly efficient approach to introduction of the single-base mutation in a plasmid containing adenovirus genome through Red recombination / 军事医学

Objective To construct a highly efficient approach to the introduction of the single-base mutation in a plasmid containing the adenovirus whole genome larger than 40 kb.Methods The target DNA with a mutation site was achieved by over-lapping PCR.The large plasmid with adenovirus genome and target DNA were co-transformed into Escherichia coli strain DY330 carrying a high rate Red recombination system.The positive clone was selected via colony PCR in combination with enzyme identification.The site-mutation large plasmid was transformed into E.coli strain DH10B in which the backbone of the large plasmid remained was stable.Results Two mutations were continuously introduced into the adenovirus genome,the location of which was pos.9171 and pos.24410 respectively.The integrality and stability of the plasmid backbone were verified by enzyme cutting identification.The two mutations on the plasmid were verified by DNA sequencing.Conclusion An efficient approach to the introduction of the single-base mutation in positions 9171 and 24410 from the adenovirus genome which was integrated into a plasmid is successfully established.The positive selection efficiency ranges from 5%to 15%.The construction of the approach will facilitate the study of adenovirus infection mechanism.

Combination of everolimus and MK-2206 for synergistic inhibition of hepatocarcinoma cell proliferation / 中国药理学与毒理学杂志

OBJECTIVE To examine the synergistic inhibiory effect of combination of mammalian target of sirolimus (Rapamycin) (mTOR) inhibitor everolimus and AKT inhibitor MK-2206 on hepatocar-cinoma cell proliferation. METHODS HepG2 and BEL-7402 cells were treated with sirolimus and evero-limus alone for 0, 1, 3, 6, 12 and 24 h or in combination with insulin-like growth factor 1 receptor (IGF-1R) inhibitor NVP-AEW541 or AKT inhibitor MK2206 for 24 h. p70S6K and AKT kinase activityies were detected by Western blotting. Plate clone formation assay and CCK8 assay were used to detect the growth and proliferation of hepatocarcinoma cells treated with everolimus and MK2206 alone or in combi-nation. RESULTS Sirolimus and everolimus inhibited p70S6K activity while causing feedback activa-tion of AKT kinase activity at different time points (P<0.01). NVP-AEW541 and MK-2206 could inhibit AKT kinase feedback activation by everolimus (P<0.05). Colony formation of hepatocarcinoma cells treated with everolimus and MK-2206 in combination was significantly inhibited compared with everolimus or MK-2206 alone (P<0.01). Everolimus and MK-2206 in combination inhibited the proliferation rate of two types of hepatocarcinoma cancer cells by more than 45％ compared with everolimus used alone (P<0.01). CONCLUSION The resistance of sirolimus and its derivatives in hepatocellular carcinoma cells may be achieved throngh the feedback-activated PI3K/AKT pathway, and the combination therapy can synergistically inhibit the growth and proliferation of hepatocarcinoma cells.

Gene fusion of egfp & kan and recombinant plasmid construction by red mediated in vivo homologous recombination / 生物工程学报

Recombineering, a new genetic engineering technology based on high efficiency in vivo homologous recombination, can be used in target DNA knock-in, knock-out and gene cloning. In the process of gene subcloning mediated by Recombineering technique, high-quality target DNA fragments were difficult to obtain using in vitro overlapping PCR,therefore the efficiency of in vivo homologous recombination was severely interrupted. To solve this problem, some technology improvements have been established based on the principle of Red recombinases. The PCR DNA fragments of egfp and kan genes with complementary sequences on the end of each fragment were co-introduced into a pcDNA3.1 vector and Red recombinases containing E. coli DY331 host cells by electroporation. A recombinant plasmid pcDNA3.1-egfp-kan was screened directly by antibiotic marker. The positive rates can reach to 45%. The EGFP gene expression of pcDNA3.1-egfp-kan can be observed by transient transfection of 293 eukaryotic cells.

Gene knockout and knockin on the Escherichia coli lac operon loci using pBR322-red system / 生物工程学报

pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.

Construction of recombinant plasmid using Neo/E Technology / 生物工程学报

A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.