SummaryRNA interference (RNAi), the double-stranded RNA (dsRNA) triggered post-transcriptional gene silencing, is becoming a powerful tool for reverse genetics studies. Stable RNAi, induced by the expression of inverted repeat (IR) transgenes, has been achieved in protozoa, algae, fungi, plants, and metazoans. However, the level of gene silencing is often quite variable, depending on the type of construct, transgene copy number, site of integration, and target gene. This is a hindrance in functional genomics studies, where it is desirable to suppress target genes reliably to analyze unknown phenotypes. Consequently, we explored strategies for direct selection of effective transgenic RNAi lines in Chlamydomonas reinhardtii. We initially attempted to suppress expression of the Rubisco small subunit multigene family by placing an IR, homologous to the conserved coding sequence, in the 3¢UTR of a transgene conferring resistance to bleomycin. However, this approach was fairly inefficient at inducing RNAi as many strains displayed defective transgene integration, resulting in partial or complete deletion of the IR, or low levels of dsRNA expression, presumably due to transcriptional silencing of the integrated IR transgenes. To overcome these problems we designed a system consisting of tandem IR transgenes that consistently triggered co-silencing of a gene with a selectable RNAiinduced phenotype (encoding tryptophan synthase b subunit) and another gene of interest (encoding either Ku80, an RNA-binding protein, or a thioredoxin isoform). We anticipate that this approach will be useful for generating stable hypomorphic epi-mutants in high-throughput phenotypic screens.

Significant progress in the functional understanding of microRNAs (miRNAs) has been made in mice, but a need remains to develop efficient tools for bi-allelic knockouts of miRNA in the human genome. Transcription activator-like effector nucleases (TALENs) provide an exciting platform for targeted gene ablation in cultured human cells, but bi-allelic modifications induced by TALENs alone occur at low frequency, making screening for double knockouts a tedious task. Here, we present an approach that is highly efficient in bi-allelic miRNA ablation in the human genome by combining TALENs targeting to the miRNA seed region with a homologous recombination donor vector and a positive selection strategy. A pilot test of this approach demonstrates bi-allelic miR-21 gene disruption at high frequency (∼87%) in cultured HEK293 cells. Analysis of three independent clones showed a total loss of miR-21 expression. Phenotypical analysis revealed increased miR-21 target gene expression, reduced cell proliferation, and alterations of global miRNA expression profiles. Taken together, our study reveals a feasible and efficient approach for biallelic miRNA ablation in cultured human cells and demonstrates its usefulness in elucidating miRNA function in human cells.

DNA damage occurs as a by-product of intrinsic cellular processes, like DNA replication, or as a consequence of exposure to genotoxic agents. Organisms have evolved multiple mechanisms to avoid, tolerate, or repair DNA lesions. To gain insight into these processes, we have isolated mutants hypersensitive to DNAdamaging agents in the green alga Chlamydomonas reinhardtii. One mutant, Ble-1, showed decreased survival when it was treated with methyl methanesulfonate (MMS), bleomycin, or hydrogen peroxide (H 2 O 2 ) but behaved like the wild type when it was exposed to UVC irradiation. Ble-1 carries an extensive chromosomal deletion that includes the gene encoding cytosolic thioredoxin h1 (Trxh1). Transformation of Ble-1 with a wild-type copy of Trxh1 fully corrected the MMS hypersensitivity and partly restored the tolerance to bleomycin. Trxh1 also complemented a defect in the repair of MMS-induced DNA strand breaks and alkali-labile sites. In addition, a Trxh1-␤-glucuronidase fusion protein translocated to the nucleus in response to treatment with MMS. However, somewhat surprisingly, Trxh1 failed to correct the Ble-1 hypersensitivity to H 2 O 2 . Moreover, Trxh1 suppression by RNA interference in a wild-type strain resulted in enhanced sensitivity to MMS and DNA repair defects but no increased cytotoxicity to H 2 O 2 . Thioredoxins have been implicated in oxidative-stress responses in many organisms. Yet our results indicate a specific role of Chlamydomonas Trxh1 in the repair of MMS-induced DNA damage, whereas it is dispensable for the response to H 2 O 2 . These observations also suggest functional specialization among cytosolic thioredoxins since another Chlamydomonas isoform (Trxh2) does not compensate for the lack of Trxh1.Genome integrity and stability, key components in the survival of an organism, are constantly threatened by DNA damage. Endogenous sources of DNA lesions include, among others, replication errors, spontaneous depurination, and alterations caused by reactive oxygen species (ROS) generated during normal metabolism (83). DNA damage can also result from exposure to environmental agents such as UV light, ionizing radiation, and chemical mutagens, including methyl methanesulfonate (MMS), bleomycin, and H 2 O 2 (70). Many DNA lesions, if left unrepaired, can lead to mutations, chromosomal aberrations, aneuploidy, or cell death (22, 58).A complex cellular system composed of an intricate network of surveillance and repair pathways has evolved to monitor and mend DNA damage. DNA lesions are detected by molecular sensors that signal the delay or arrest of cell cycle progression as well as an array of transcriptional and DNA repair responses (83). DNA repair responses include direct repair, base excision repair (BER), nucleotide excision repair (NER), mismatch repair, and DNA double-strand break (DSB) repair (22,58). Base excision repairs oxidized, alkylated (usually methylated), or deaminated bases and single-strand breaks (SSBs) (48), whereas NER is the major repair system for removing bulky, helix-di...

Background: Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. Results: We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions.

BackgroundA number of integrase defective lentiviral (IDLV) packaging systems have been developed to produce integration deficient lentiviruses for gene delivery and epichromosomal expression. However, despite their growing demand, a comparative study to systemically evaluate the performance efficiency of different mutants on virus packaging and gene expression has not been done.ResultsSite-directed mutagenesis was used to generate five integrasedeficient mutants for non-integrative lentiviral packaging (NILVP). The five mutants were then individually incorporated to make different integrase defective lentivirus plasmid packaging mix, keeping other packaging factors constant. CD511B-1, a lentivectorexpressing GFP from an EF1 promoter, was packaged with each of the five different lentivirus packaging mix to make pseudotypedviral particles. The performance and packaging efficiency of each of the integrase deficient mutants was evaluated based on GFP expression in HT1080 cells, while the wild type lentivirus packaging mix was used as a control. Of the five integrase mutant candidates, one with the highestGFP transgene expression level was chosen for further characterization. The non-integrative nature of this candidate was confirmed by quantitative polymerase chain reaction and characterized using both dividing and non-dividing cells. Finally, a detailed standard protocol for NILVP using this integrase defective mutant was developed.ConclusionsAn efficient lentiviral packaging system for producing on-integrative lentivirus was established. This system is compatible with most existing lentivectors and can be used to transduce both dividing and non-dividing cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-016-0044-z) contains supplementary material, which is available to authorized users.

Reverse genetic approaches have become invaluable tools to tap into the wealth of information provided by sequenced genomes. In 2007, sequencing of the Chlamydomonas reinhardtii genome was completed, and with this an increased demand for the development of reverse genetic strategies for gene analysis. In a variety of organisms, including Chlamydomonas, inverted repeat transgenes have been used to produce strains silenced for a specific gene due to the production of double stranded RNA (dsRNA). Here, we describe a tandem inverted repeat system designed to overcome some of the typical challenges that arise when transgenes are used to trigger gene silencing including the lack of a screenable phenotype, unpredictable levels of silencing, silencing of the transgene itself and thus loss of target gene silencing, and finally silencing of unintended genes (off-target genes). The described strategy allows selection of target gene silencing by inducing co-silencing of the target gene and a gene, MAA7, silencing of which produces a selectable RNAi-induced phenotype. This selection, therefore, precludes extensive molecular screening for transgenic strains exhibiting target gene silencing, and also ensures heritable silencing through many generations.

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