Zinc Finger Nuclease Technology Research Articles

Review of knockout technology approaches in bacterial drug resistance research.

Gene knockout is a widely used method in biology for investigating gene function. Several technologies are available for gene knockout, including zinc-finger nuclease technology (ZFN), suicide plasmid vector systems, transcription activator-like effector protein nuclease technology (TALEN), Red homologous recombination technology, CRISPR/Cas, and others. Of these, Red homologous recombination technology, CRISPR/Cas9 technology, and suicide plasmid vector systems have been the most extensively used for knocking out bacterial drug resistance genes. These three technologies have been shown to yield significant results in researching bacterial gene functions in numerous studies. This study provides an overview of current gene knockout methods that are effective for genetic drug resistance testing in bacteria. The study aims to serve as a reference for selecting appropriate techniques.

Research progress in mitochondrial gene editing technology.

Mitochondrial DNA (mtDNA) mutations result in a variety of genetic diseases. As an emerging therapeutic method, mtDNA editing technology recognizes targets more based on the protein and less on the nucleic acid. Although the protein recognition type mtDNA editing technology represented by zinc finger nuclease technology, transcription activator like effector nuclease technology and base editing technology has made some progress, the disadvantages of complex recognition sequence design hinder further popularization. Gene editing based on nucleic acid recognition by the CRISPR system shows superiority due to the simple structure, easy design and modification. However, the lack of effective means to deliver nucleic acids into mitochondria limits application in the field of mtDNA editing. With the advances in the study of endogenous and exogenous import pathways and the deepening understanding of DNA repair mechanisms, growing evidence shows the feasibility of nucleic acid delivery and the broad application prospects of nucleic acid recognition type mtDNA editing technology. Based on the classification of recognition elements, this article summarizes the current principles and development of mitochondrial gene editing technology, and discusses its application prospects.

Improvement of Workflows and Assay Reproducibility by The Introduction of "Assay-Ready" Culturing of MDCK Cells for Transport Studies.

In previous studies, we established and validated three Madin Darby Canine Kidney MDCKII cell lines, recombinantly modified with zinc finger nuclease (ZFN) technology. Here, we investigated the applicability of seeding these three canine P-gp deficient MDCK_ZFN cell lines, directly from frozen cryopreserved stocks without previous cultivation for efflux transporter and permeability studies. This technique is referred to as "assay-ready" and allows for highly standardized conduction of cell-based assays and shorter cultivation cycles. To obtain a rapid fitness of the cells for that purpose, a very gentle freezing and thawing protocol was applied. Assay-ready MDCK_ZFN cells were tested in bi-directional transport studies and compared to their traditionally cultured counterparts. Long-term performance robustness, human effective intestinal permeability (Peff) predictability and batch to batch variability were assessed. Efflux ratios (ER) and apparent permeability (Papp) results were highly comparable between assay-ready and standard cultured cell lines with R2 values of 0.96 or higher. Papp to Peff correlations obtained from passive permeability with non-transfected cells were comparable independent of the cultivation regime. Long-term evaluation revealed robust performance of assay-ready cells and reduced data variability of reference compounds in 75% of cases compared to standard cultured MDCK_ZFN cells. Assay-ready methodology for handling MDCK_ZFN cells allows more flexibility in assay planning and reduces performance fluctuations in assays caused by cell aging. Therefore, the assay-ready principle has proven superior over conventional cultivation for MDCK_ZFN cells and is considered as a key technology to optimize processes with other cellular systems.

Research and prospect of CRISPR system in marine biological system

Gene editing is a widely used and promising technology in biology field. Its ability to precisely edit the endogenous genes of organisms is a major boost to the research process in the biological sciences. Among these gene editing technologies, CRISPR is a more effective system and it is in a wider range of applications. The CRISPR system has advantages over Zinc finger nuclease (ZFNs) technology and transcription activator-like effector nuclease (TALENs) technology, including higher efficiency, gentler construction and a decreased risk of off-target effects. Currently, the treatment of diseases, the creation of animal models, and the genetic breeding of plants all make extensive use of CRISPR technology. Despite its wide request in model construction, its application in marine organisms is just beginning. This review systematically introduces the classification, development of gene editing technology, and summarizes the current status and development prospects of the application of CRISPR system in marine organisms.

Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells

BackgroundRedirection of natural killer (NK) cells with chimeric antigen receptors (CAR) is attractive in developing off-the-shelf CAR therapeutics for cancer treatment. However, the site-specific integration of a CAR gene into NK cells remains challenging.MethodsIn the present study, we genetically modified human induced pluripotent stem cells (iPSCs) with a zinc finger nuclease (ZFN) technology to introduce a cDNA encoding an anti-EpCAM CAR into the adeno-associated virus integration site 1, a “safe harbour” for transgene insertion into human genome, and next differentiated the modified iPSCs into CAR-expressing iNK cells.ResultsWe detected the targeted integration in 4 out of 5 selected iPSC clones, 3 of which were biallelically modified. Southern blotting analysis revealed no random integration events. iNK cells were successfully derived from the modified iPSCs with a 47-day protocol, which were morphologically similar to peripheral blood NK cells, displayed NK phenotype (CD56+CD3-), and expressed NK receptors. The CAR expression of the iPSC-derived NK cells was confirmed with RT-PCR and flow cytometry analysis. In vitro cytotoxicity assay further confirmed their lytic activity against NK cell-resistant, EpCAM-positive cancer cells, but not to EpCAM-positive normal cells, demonstrating the retained tolerability of the CAR-iNK cells towards normal cells.ConclusionLooking ahead, the modified iPSCs generated in the current study hold a great potential as a practically unlimited source to generate anti-EpCAM CAR iNK cells.

Time course of ventilatory failure in a novel rat model of Pompe Disease

Pompe disease (PD) is caused by mutations in the gene encoding the enzyme acid-α-glucosidase (GAA). Since GAA acts to degrade lysosomal glycogen, reduced or loss of enzyme activity leads to glycogen accumulation and neuromuscular impairments. Respiratory dysfunction and ventilator-dependence is common in both early- and late-onset PD. Our group has created a rat model of PD using Zinc Finger Nuclease (ZFN) technology. The goal of the current study was to characterize ventilation and metabolism in unrestrained ZFN Pompe rats using whole body plethysmography. Data were collected during a normoxic baseline period, acute exposures to hypoxia (FiO2=10.5%), hypercapnia (FiCO2=7%), and combined hypercapnic-hypoxic challenge intended to produce a maximal ventilatory drive. ZFN Pompe rats and age-matched, wild type (WT) controls were studied at age 3, 6, and 10-11 months to assess ventilation throughout disease progression. Both male and female 3 month old ZFN Pompe rats hyperventilated (greater VE/VCO2) due to greater tidal volume compared to WT rats. However, as the ZFN Pompe rats aged, the ventilatory phenotype changed such that by 10-11 months they showed significant hypoventilation as well as a blunted ability to increase ventilation in response to respiratory challenge relative to WT controls. Histological data from these rats, as well as prior data from the Gaa-/- mouse model of PD, show that respiratory neuromuscular pathology is present at a very young age. Accordingly, the current ventilatory data suggest that animals with PD initially increase breathing in response to the onset of pathology, possibly representing a period of “overcompensation”. However, this is followed by respiratory failure as the respiratory neuromuscular pathology progresses towards disease end-stage.

Overview of sixteen scientific opinions on genetically modified plants obtained by new genomic techniques

RIVM was asked by EFSA in the frame of a procurement (PO/EFSA/GMO/2020/01) to producean overview of 16 scientific opinions published by European competent authorities and national institutions since 2012 ongenetically modified plants obtained by new genomic techniques (NGTs). The 16 opinions were provided by the European Commission in the frame of a mandate asking EFSA to provide an overview on the risk assessment of plants developed through NGTsbased on its own previous and current work and on work carried out at national level. NGTs are defined as techniques capable to change the genetic material of an organism and that have emerged or have been developed since the adoption of the GMO legislation in 2001. Based on this definition for NGTs provided by EC, the following NGTs as described in a report of the Joint Research Council on new plant breeding techniques, published in 2011, were considered: (1) Zinc finger nuclease technology, (defined more broadly as site-directed nuclease technology); (2) oligonucleotide directed mutagenesis; (3) cisgenesis and intragenesis; (4) RNA-dependent DNA methylation; (5) grafting (on genetically modified rootstock); (6) reverse breeding; (7) agro-infiltration and (8) synthetic genomics.The current report presents the information on the defined NGTsmethod descriptionand on the risk assessment of plants developed through these NGTs, as extracted from the 16opinions. In order to extract the relevant information a baseline descriptionfor each NGT was set and inclusion and exclusion criteria for information extraction were defined. Most (14 out of 16) opinions discuss the SDN technology, whereas no opinion contains information on synthetic genomics. A new NGT, base-editing, is described in four opinions and therefore information on this technique is also presented.As specified by the procurement, no critical appraisal onthe information extracted from the scientific opinions was performed.

ZFN-mediated in vivo gene editing in hepatocytes leads to supraphysiologic α-Gal A activity and effective substrate reduction in Fabry mice

Fabry disease, a lysosomal storage disorder resulting from thedeficient activity of α-galactosidase A (α-Gal A), is characterized by cardiac, renal, and/or cerebrovascular disease due to progressive accumulation of the enzyme's substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). We report here the preclinical evaluation of liver-targeted invivo genome editing using zinc-finger nuclease (ZFN) technology to insert the human α-galactosidase A (hGLA) cDNA into the albumin "safe harbor" locus of Fabry mice, thereby generating an albumin-α-Gal A fusion protein. The mature α-Gal A protein is secreted into the circulation for subsequent mannose-6-phosphate receptor-mediated tissue uptake. Donor vector optimization studies showed that replacing the hGLA cDNA signal peptide sequence with that of human iduronate 2-sulfatase (IDS) achieved higher transgene expression. Intravenous adeno-associated virus (AAV) 2/8-mediated co-delivery of the IDS-hGLA donor and ZFNs targeting the albumin locus resulted in continuous, supraphysiological plasma and tissue α-Gal A activities, which essentially normalized Gb3 and Lyso-Gb3 levels in key tissues of pathology. Notably, this was achieved with <10% of hepatocytes being edited to express hGLA, occurring mostly via non-homologous end joining (NHEJ) rather than homology-directed repair (HDR). These studies indicate that ZFN-mediated invivo genome editing has the potential to be an effective one-time therapy for Fabry disease.

Strategies for mitochondrial gene editing.

Mitochondria, as the energy factory of cells, participate in metabolism processes and play a critical role in the maintenance of human life activities. Mitochondria belong to semi-automatic organelles, which have their own genome different from nuclear genome. Mitochondrial DNA (mtDNA) mutations can cause a series of diseases and threaten human health. However, an effective approach to edit mitochondrial DNA, though long-desired, is lacking. In recent years, gene editing technologies, represented by restriction endonucleases (RE) technology, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, CRISPR system and pAgo-based system have been comprehensively explored, but the application of these technologies in mitochondrial gene editing is still to be explored and optimized. The present study highlights the progress and limitations of current mitochondrial gene editing technologies and approaches, and provides insights for development of novel strategies for future attempts.

Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes.

Background and purpose:The study was launched to use zinc finger nuclease (ZFN) technology to disrupt the cholera toxin gene (ctxA) for inhibiting CT toxin production in Vibrio cholera (V. cholera).Experimental approach:An engineered ZFN was designed to target the catalytic site of the ctxA gene. The coding sequence of ZFN was cloned to pKD46, pTZ57R T/A vector, and E2-crimson plasmid and transformed to Escherichia coli (E. coli) Top10 and V. cholera. The efficiency of ZFN was evaluated by colony counting.Findings/Results:No expression was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting in transformed E. coli. The ctxA gene sequencing did not show any mutation. Polymerase chain reaction on pKD46-ZFN plasmid had negative results. Transformation of E. coli Top10 with T/A vectors containing whole ZFN sequence led to 7 colonies all of which contained bacteria with self-ligated vector. Transformation with left array ZFN led to 24 colonies of which 6 contained bacteria with self-ligated vector and 18 of them contained bacteria with vector/left array. Transformation of V. cholera with E2-crimson vectors containing whole ZFN did not produce any colonies. Transformation with left array vectors led to 17 colonies containing bacteria with vector/left array. Left array protein band was captured using western blot assay.Conclusions and implications:ZFN might have off target on bacterial genome causing lethal double-strand DNA break due to lack of non-homologous end joining (NHEJ) mechanism. It is recommended to develop ZFNs against bacterial genes, engineered packaging host with NHEJ repair system is essential.

Identification of genes related to skeletal muscle growth and development by integrated analysis of transcriptome and proteome in myostatin-edited Meishan pigs

Embryonic development of skeletal muscle is a complex process that is important to the growth of skeletal muscle after birth. However, the mechanisms by which skeletal muscle growth and development in embryonic phase remain unclear. We have previously produced myostatin-knockout (MKO) Meishan pigs with double-muscle (DM) phenotype via zinc finger nucleases (ZFN) technology. To further investigate the molecular mechanisms involved in skeletal muscle growth and development, in this study, we performed an integrated analysis of transcriptome and proteome in longissimus dorsi muscle from myostatin wild type (MWT) and MKO pigs on 65 days post coitus. Results showed that, compared with MWT group, there were 438 differentially expressed genes (DEGs) and 66 differentially expressed proteins (DEPs) in MKO group. These DEGs and DEPs were mainly enriched in signaling pathways that are involved in skeletal muscle growth and development, glucose metabolism and apoptosis. Furthermore, we identified two key genes, Troponin T 1 (TNNT1) and Myosin regulatory light chain 9 (MYL9), which showed significant changes in both mRNA and protein levels with the similar changing trends in MKO group. It is thus speculated that TNNT1 and MYL9 may play an important role in skeletal muscle growth and development. SignificanceOur study analyzed some important regulatory genes and proteins during skeletal muscle growth and development, our results provided (1) a new insight to further understanding of the molecular mechanisms by which growth and development are regulated in porcine skeletal muscle, and (2) some possible molecular makers for improvement of meat quality in the animal husbandry and diagnosis of human muscle diseases in medicine.

Generation of Transmission-Competent Human Malaria Parasites with Chromosomally-Integrated Fluorescent Reporters

Malaria parasites have a complex life cycle that includes specialized stages for transmission between their mosquito and human hosts. These stages are an understudied part of the lifecycle yet targeting them is an essential component of the effort to shrink the malaria map. The human parasite Plasmodium falciparum is responsible for the majority of deaths due to malaria. Our goal was to generate transgenic P. falciparum lines that could complete the lifecycle and produce fluorescent transmission stages for more in-depth and high-throughput studies. Using zinc-finger nuclease technology to engineer an integration site, we generated three transgenic P. falciparum lines in which tdtomato or gfp were stably integrated into the genome. Expression was driven by either stage-specific peg4 and csp promoters or the constitutive ef1a promoter. Phenotypic characterization of these lines demonstrates that they complete the life cycle with high infection rates and give rise to fluorescent mosquito stages. The transmission stages are sufficiently bright for intra-vital imaging, flow cytometry and scalable screening of chemical inhibitors and inhibitory antibodies.

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for invitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

PKC\u03b6 facilitates lymphatic metastatic spread of prostate cancer cells in a mice xenograft model

Prostate cancer disseminates primarily into the adjacent lymph nodes, which is related to a poor outcome. Atypical protein kinase C ζ (PKCζ) is highly expressed in aggressive prostate cancer and correlates with Gleason score, clinical stage, and poor prognosis. Here, we report the molecular mechanisms of PKCζ in lymphatic metastasis during prostate cancer progression. Using zinc-finger nuclease technology or PKCζ shRNA lentiviral particles, and orthotopic mouse xenografts, we show that PKCζ-knockout or knockdown from aggressive prostate cancer (PC3 and PC3U) cells, decreasesd tumor growth and lymphatic metastasis in vivo. Intriguingly, PKCζ-knockout or knockdown impaired the activation of AKT, ERK, and NF-κB signaling in prostate cancer cells, thereby impairing the expression of lymphangiogenic factors and macrophage recruitment, resulting in aberrant lymphangiogenesis. Moreover, PKCζ regulated the expression of hyaluronan synthase enzymes, which is important for hyaluronan-mediated lymphatic drainage and tumor dissemination. Thus, PKCζ plays a crucial oncogenic role in the lymphatic metastasis of prostate cancer and is predicted to be a novel therapeutic target for prostate cancer.

SU104 - EXPERIMENTAL MODEL OF DISRUPTED IN SCHIZOPHRENIA 1 GENE IN A STEM CELL BASED NEURONAL MODEL

Background Induced Pluripotent Stem Cells (IPSCs) have the capability to differentiate into any kind of cell types, such as Neuronal Progenitor Cells (NPCs), and then to mature neurons. By this new approach we can investigate neuronal differentiation processes or the role of specific genes using genome editing techniques (e.g., ZFN, TALEN, or CRISPR). For example, the neuron-specific roles of the disrupted in schizophrenia 1 (DISC1) can be studied in a simple, Petri-dish based model. The DISC1gene has been intensively studied over the last decades after the description of a balanced translocation of t(1;11)(q42.1;q14.3) in a multigenerational Scottish family with several cases of psychosis and affective disorders, followed by the identification of a frameshift DISC1 mutation in an American family affected with schizophrenia. Previous molecular biological experiments have shown that DISC1 scaffold protein can affect neuronal proliferation and migration, and synapse maintenance. Animal model findings also pointed to its role in hippocampal neurogenesis, which is a crucial neuronal mechanism affected in several psychiatric disorders. Methods We aimed to characterize the possible roles of DISC1 in vitro neuronal cell culture. We applied an isogenic IPSC line in a model of hippocampal dentate gyrus neurogenesis, given that impaired hippocampal neurogenesis has been implicated in the pathogenesis of both schizophrenia and affective disorders. IPSC line XCL1 was engineered using zinc finger nuclease technology to produce an isogenic DISC1 exon-2 biallelic knockout cell line (XCL1 DISC1 exon-2 KO/KO). The locus of the genetic engineering was selected because of the frameshift mutation. The isogenic IPSC pair was characterized in terms of pluripotency and spontaneous differentiation potential. Following this, IPSC lines were differentiated into neuronal lineages using established protocols to generate hippocampal dentate gyrus granule cells. The dentate gyrus protocol is founded on dual SMAD inhibition, Wnt- and SHH inhibition, after generating free-floated embryoid bodies from pluripotent stem cells, which results in forebrain NPCs. Results We have successfully derived and characterized NPCs and then matured neurons from human IPSC XCL1 and deletion pair, which were differentiated according to the DG PROX1 protocol. There was no significant difference at the spontaneous differentiation potential between the isogenic IPSC XCL1 lines. The immunofluorescence staining showed that NPCs express Nestin and Sox2. This intermediate cell type can be further differentiated into PROX1 and MAP2 positive functional neurons which have dendritic spines. Till now we have not found significant differences between wild type and knock out cell lines based on induced outgrowth measurement and structure of ICC. We have found some alteration in Ca-dynamics between the two cell lines, however these measurements need further investigation. Discussion The generated neuronal progenitor cells and mature neurons carrying biallelic knockout of the DISC1 gene were amenable for assays comparing the isogenic pairs by immunofluorescence microscopy, and transcriptomics. Functional studies, such as calcium-imaging assays, single cell patch-clamp electrophysiology and real-time live cell visualization of induced neurite outgrowth are under way to further. These investigations may help to explore the role of DISC1 in hippocampal neurogenesis and the etiology of psychiatric disorders.

A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats

Tobacco addiction is a chronic and relapsing disorder with an important genetic component that represents a major public health issue. Meta-analysis of large-scale human genome-wide association studies (GWASs) identified a frequent non-synonymous SNP in the gene coding for the α5 subunit of nicotinic acetylcholine receptors (α5SNP), which significantly increases the risk for tobacco dependence and delays smoking cessation. To dissect the neuronal mechanisms underlying the vulnerability to nicotine addiction in carriers of the α5SNP, we created rats expressing this polymorphism using zinc finger nuclease technology and evaluated their behavior under the intravenous nicotine-self-administration paradigm. The electrophysiologicalresponses of their neurons to nicotine were also evaluated. α5SNP rats self-administered more nicotine at high doses and exhibited higher nicotine-induced reinstatement of nicotine seeking than wild-type rats. Higher reinstatement was associated with altered neuronal activity in several discrete areas that are interconnected, including in the interpeduncular nucleus (IPN), aGABAergic structure that strongly expresses α5-containing nicotinic receptors. The altered reactivity of IPN neurons of α5SNP rats to nicotine was confirmed electrophysiologically. In conclusion, the α5SNP polymorphism is a major risk factor for nicotine intake at high doses and for relapse to nicotine seeking in rats, a dual effect that reflects the human condition. Our results also suggest an important role for the IPN in the higher relapse to nicotine seeking observed in α5SNP rats.

Genetically Engineered iPSC-Derived FTDP-17 MAPT Neurons Display Mutation-Specific Neurodegenerative and Neurodevelopmental Phenotypes.

SummaryTauopathies such as frontotemporal dementia (FTD) remain incurable to date, partially due to the lack of translational in vitro disease models. The MAPT gene, encoding the microtubule-associated protein tau, has been shown to play an important role in FTD pathogenesis. Therefore, we used zinc finger nucleases to introduce two MAPT mutations into healthy donor induced pluripotent stem cells (iPSCs). The IVS10+16 mutation increases the expression of 4R tau, while the P301S mutation is pro-aggregant. Whole-transcriptome analysis of MAPT IVS10+16 neurons reveals neuronal subtype differences, reduced neural progenitor proliferation potential, and aberrant WNT/SHH signaling. Notably, these neurodevelopmental phenotypes could be recapitulated in neurons from patients carrying the MAPT IVS10+16 mutation. Moreover, the additional pro-aggregant P301S mutation revealed additional phenotypes, such as an increased calcium burst frequency, reduced lysosomal acidity, tau oligomerization, and neurodegeneration. This series of iPSCs could serve as a platform to unravel a potential link between pathogenic 4R tau and FTD.

Research progress of gene editing technology CRISPR/Cas9 system in animal gene editing

Gene editing technology, from the beginning of RNA interference (RNAi) technology to efficient developed enzyme technology, has been widely used in recent years. These efficient enzyme technologies include zinc finger nuclease (ZFN) technology, transcriptional activation-like effector nuclease (TALENs) technology, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system (CRISPR/Cas9) technology.

Pronounced Hyperactivity, Cognitive Dysfunctions, and BDNF Dysregulation in Dopamine Transporter Knock-out Rats.

Dopamine (DA) controls many vital physiological functions and is critically involved in several neuropsychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder. The major function of the plasma membrane dopamine transporter (DAT) is the rapid uptake of released DA into presynaptic nerve terminals leading to control of both the extracellular levels of DA and the intracellular stores of DA. Here, we present a newly developed strain of rats in which the gene encoding DAT knockout Rats (DAT-KO) has been disrupted by using zinc finger nuclease technology. Male and female DAT-KO rats develop normally but weigh less than heterozygote and wild-type rats and demonstrate pronounced spontaneous locomotor hyperactivity. While striatal extracellular DA lifetime and concentrations are significantly increased, the total tissue content of DA is markedly decreased demonstrating the key role of DAT in the control of DA neurotransmission. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, the partial Trace Amine-Associated Receptor 1 (TAAR1) agonist RO5203648 ((S)-4-(3,4-Dichloro-phenyl)-4,5-dihydro-oxazol-2-ylamine) and haloperidol. DAT-KO rats also demonstrate a deficit in working memory and sensorimotor gating tests, less propensity to develop obsessive behaviors and show strong dysregulation in frontostriatal BDNF function. DAT-KO rats could provide a novel translational model for human diseases involving aberrant DA function and/or mutations affecting DAT or related regulatory mechanisms.SIGNIFICANCE STATEMENT Here, we present a newly developed strain of rats in which the gene encoding the dopamine transporter (DAT) has been disrupted (Dopamine Transporter Knockout rats [DAT-KO rats]). DAT-KO rats display functional hyperdopaminergia accompanied by pronounced spontaneous locomotor hyperactivity. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, and a few other compounds exerting inhibitory action on dopamine-dependent hyperactivity. DAT-KO rats also demonstrate cognitive deficits in working memory and sensorimotor gating tests, less propensity to develop compulsive behaviors, and strong dysregulation in frontostriatal BDNF function. These observations highlight the key role of DAT in the control of brain dopaminergic transmission. DAT-KO rats could provide a novel translational model for human diseases involving aberrant dopamine functions.

Enhanced Manipulation of Human Mitochondrial DNA Heteroplasmy In Vitro Using Tunable mtZFN Technology.

As a platform capable of mtDNA heteroplasmy manipulation, mitochondrially targeted zinc-finger nuclease (mtZFN) technology holds significant potential for the future of mitochondrial genome engineering, in both laboratory and clinic. Recent work highlights the importance of finely controlled mtZFN levels in mitochondria, permitting far greater mtDNA heteroplasmy modification efficiencies than observed in early applications. An initial approach, differential fluorescence-activated cell sorting (dFACS), allowing selection of transfected cells expressing various levels of mtZFN, demonstrated improved heteroplasmy modification. A further, key optimization has been the use of an engineered hammerhead ribozyme as a means for dynamic regulation of mtZFN expression, which has allowed the development of a unique isogenic cellular model of mitochondrial dysfunction arising from mutations in mtDNA, known as mTUNE. Protocols detailing these transformative optimizations are described in this chapter.