The first report on successful enhanced gene targeting


The first report on successful enhanced gene targeting

by impairing NHEJ was in Kluyveromyces lactis (6). Deletion of KlKu80, one of the key factors in NHEJ, increased targeting efficiency even for homologous flanking regions spanning 100 bp (6). Since then, the NHEJ pathway has been impaired to increase HR frequency in many other fungi (8–11). In Neurospora crassa, impairment of the NHEJ pathway by deletion of mus-51 (Ku70) and mus-52 (Ku80) resulted in marked increases in HR frequency in comparison to wild-type controls (8). Moreover, a recent study demonstrated that mus-53 (homolog of human lig4) is specific for NHEJ and functions in the final step of NHEJ (12). Disruption of mus-53 resulted in an HR frequency of 100%. Similar results were obtained in LIGD-deficient Aspergillus oryzae (13). However, 100% HR frequency was not achieved in all disrupted loci. In the dermatophyte T. HM781-36B clinical trial mentagrophytes, a single

trial has been performed on increasing gene targeting efficiency by HR (14). However, not all integration events occur in the HR pathway. To obtain a much higher homologous recombination frequency, we studied the HR pathway in a Lig4-null mutant of T. mentagrophytes. In this study, we isolated a lig4 ortholog in T. mentagrophytes (TmLIG4). Evaluation of HI frequency in the TmLIG4Δ disruptant was observed at four different loci. Strains used in this study are listed in Table 1. TIMM2789 was used as the recipient strain to produce TMLIG4 defective mutants. It was maintained on solid SDA at 28°C. Transformants were maintained on SDA supplemented with either 100 μg/mL G418 or 100 μg/mL hygromycin B. Conidial formation of each T. mentagrophytes strain was induced using modified

1/10 SDA (16) supplemented with appropriate antibiotics. For total DNA extraction, growing mycelia from each T. mentagrophytes Oxymatrine strain were collected after incubation for 5 days at 28°C on SDA supplemented with 500 μg/mL cycloheximide, 50 μg/mL chloramphenicol and 100 μg/mL G418 or hygromycin. Aspergillus minimal broth (17) supplemented with 50 μg/mL chloramphenicol was used to obtain mycelia for total RNA extraction with an RNeasy Plant Mini Kit (Qiagen, Gaithersburg, MD, USA). EHA105 was maintained on solid 2×YT medium (1.6%[w/v] tryptone, 1.0%[w/v] yeast extract, 0.5%[w/v] NaCl and 1.5%[w/v] agar) supplemented with 50 μg/mL rifampicin and 25 μg/mL chloramphenicol at 28°C. For routine cloning, DH5α (Nippon Gene, Toyama, Japan) was used. Based on the amino acid sequences of four fungal Lig4, a pair of degenerate primers was designed (MP-F1 and MP-R1) and used to amplify an internal fragment by PCR. The amplified fragment was sequenced, and both ends extended using a Genome Walker kit (Clontech, Palo Alto, CA, USA). To amplify both ends, a set of six specific primers were designed. The 3′ end of the TmLIG4 ORF was determined by amplification of the partial cDNA fragment with 3′ rapid amplification of cDNA ends (Invitrogen, Carlsbad, CA, USA).

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