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Genetic Engineering Publications - GEG Tech top picks
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BigField GEG Tech's insight:
In this study, the authors design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 to generate reporter systems while retaining pluripotent characteristics. They demonstrate the possibility to use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. The results provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays. 
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PhiC31 integrase-mediated gene delivery has been extensively used in gene therapy and animal transgenesis. However, random integration events are observed in phiC31-mediated integration in different types of mammalian cells; as a result, the efficiencies of pseudo attP site integration and evaluation of site-specific integration are compromised. To improve this system, we used an attB-TK fusion gene as a negative selection marker, thereby eliminating random integration during phiC31-mediated transfection. We also excised the selection system and plasmid bacterial backbone by using two other site-specific recombinases, Cre and Dre. Thus, we generated clean transgenic bovine fetal fibroblast cells free of selectable marker and plasmid bacterial backbone. These clean cells were used as donor nuclei for somatic cell nuclear transfer (SCNT), indicating a similar developmental competence of SCNT embryos to that of non-transgenic cells. Therefore, the present gene delivery system facilitated the development of gene therapy and agricultural biotechnology.
BigField GEG Tech's insight:
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BigField GEG Tech's insight:
In this study, the authors explored building iKO hPSC lines by combining CRISPR/Cas9-mediated genome editing with the Flp/FRT and Cre/LoxP system. In this way, they developed a strategy to simultaneously insert an activity-controllable recombinase-expressing cassette and remove the drug-resistance gene, thus speeding up the generation of iKO hPSC lines. This two-step strategy was used to establish human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) lines.
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PLOS ONE: an inclusive, peer-reviewed, open-access resource from the PUBLIC LIBRARY OF SCIENCE. Reports of well-performed scientific studies from all disciplines freely available to the whole world.
BigField GEG Tech's insight:
Genetic engineering of iPs invloving 3 site specific recombinases. PhiC31 integrase was used to mediate initial placement of a single copy of a reprogramming plasmid into the genome at a safe location. A second phage integrase, Bxb1, was used to place the full-length dystrophin coding sequence into the same location, and Cre resolvase was utilized to excise unwanted sequences.
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In this study two strategies were used to increase the precise genetic modification in rats. Scr7, a DNA ligase IV inhibitor, first identified as an anti-cancer compound, and considered as a potential NHEJ inhibitor, was used to increase the HR-mediated precise genetic modification. Meanwhile, the Cas9 protein instead of mRNA was used to save the mRNA to protein translation step to improve the precise modification efficiency. The Fabp2 and Dbndd1 loci were selected to knockin Cre and CreERT2, respectively. These results showed that both Scr7 and Cas9 protein can increase the precise modification.