Gene Targeting Methods Research Articles

Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice

CRISPR/Cas9 technology is a highly promising genome editing tool in the mouse, potentially overcoming the costs and time required for more traditional gene targeting methods in embryonic stem (ES) cells. Recently, compared to the wildtype nuclease, paired Cas9 nickase (Cas9n) combined with single guide RNA (sgRNA) molecules has been found to enhance the specificity of genome editing while reducing off-target effects. Paired Cas9n has been shown to be as efficient as Cas9 for generating insertion and deletion (indel) mutations by non-homologous end joining and targeted deletion in the genome. However, an efficient and reliable approach to the insertion of loxP sites flanking critical exon(s) to create a conditional allele of a target gene remains an elusive goal. In this study, we microinjected Cas9n RNA with sgRNAs together with a single DNA template encoding two loxP sites flanking (floxing) exon 2 of the isoprenoid synthase containing domain (Ispd) into the pronucleus and cytoplasm of C57BL/6NCr one-cell stage zygotes. After surgical transfer, one F0 mouse expressing a conditional allele was produced (at a frequency of ∼8% of live pups born). The floxed allele was transmitted through the germline to F1 progeny, and could be successfully recombined using Cre recombinase. This study indicates that conditional targeting can be accomplished effectively using paired Cas9n and a single DNA template.

220 LENGTH OF DONOR DNA HOMOLOGY TO FACILITATE BI-ALLELIC GENE TARGETING DURING TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-MEDIATED GENE TARGETING

Gene editing techniques are increasing in their availability and ease of use. These techniques are an attractive alternative to conventional gene targeting methods via homologous recombination due to the ease of use and the efficiency of targeting. We have previously produced CMP-N-acetylneuraminic acid hydroxylase (CMAH) knockout (KO) pigs in a Minnesota miniature pig genetic background. These pigs were generated using zinc-finger nucleases (ZFN) in combination with donor DNA containing a total length of homology of 1600 bp (800-bp homology on each arm). A second KO was desired in the CMAH –/– genetic background, GGTA1. We used this opportunity to evaluate the effect of total donor homology on transcription activator-like effector nuclease (TALEN) mediated bi-allelic modification. Donor DNA was designed to symmetrically flank the predicted TALEN cleavage site in GGTA1 and was varied in length of total homology (40, 100, 200, 460, 890, and 1800 bp). The TALEN constructs and donor DNA were introduced into fibroblast cells by a lipid-based transfection method. Each donor DNA was co-transfected with a pair of TALEN constructs. In addition, the TALEN pair alone and the longest donor DNA alone were separately transfected to evaluate error-prone non-homologous end joining (NHEJ) repair or background HR, respectively. Five days post transfection, cells were evaluated for the ablation of the α-1,3 gal epitope, which results from bi-allelic disruption of GGTA1. Lectin IB-4 (FITC labeled) was used to detect the presence/absence of the epitope. If a bi-allelic modification to the GGTA1 gene was accomplished, specific binding of the lectin would not occur; therefore, a lack of FITC was evaluated. Donor DNA alone did not result in detectable bi-allelic conversion of GGTA1. As the length of donor DNA increased, the bi-allelic disruption of GGTA1 increased from 0.5% (no donor DNA) to a maximum of 3% (1800 bp). Inclusion of homologous donor DNA in TALEN transfections facilitates HR that competes with error-free NHEJ. The use of donor DNA with TALEN therefore can provide for increased bi-allelic disruption and predictability in the sequence of the resulting mutation.

224 CONSTRUCTION OF GENE TARGETING VECTORS FOR RAG-1 AND RAG-2

Gene targeting in mammalian cells has become a routine technique and is currently used to study gene function, create biomedical models, and generate potential tissue sources for xenotransplantation. Severe combined immunodeficiency (SCID) is a condition characterised by the absence of T cells and a lack of B cell function. Severe combined immunodeficiency affects ~1 out of every 100 000 infants. Autosomal recessive SCID can occur due to a mutation within the recombination activating genes (RAG-1/RAG-2) that play a role in recombination of immunoglobulins and T-cell receptors. Gene targeting has been used to create mouse models to study the effects of a RAG-1 or RAG-2 deficiency on the immune system. In 1992, Mombaerts et al. generated a homozygous mouse model of RAG-1 deficiency, whereas Shinkai et al. generated a homozygous mouse model of RAG-2. Both models resulted in the absence of mature T or B lymphocytes; which was concluded to be due to the lack of the ability to initiate the V(D)J recombination process. Because of the anatomical and physiological similarities between humans and pigs, a swine model of both RAG-1 and RAG-2 deficiency would have utility. We hypothesise that disruption of RAG-1, RAG-2, or both in swine will result in a SCID phenotype. A first step in the creation of a swine SCID model is to assemble targeting vectors. The objective of this work was to construct targeting vectors. To accomplish this goal, genomic DNA from porcine fetal fibroblasts was used to amplify a 6840-bp PCR product including the porcine RAG-1 gene. This fragment was cloned into TOPO pCR-XL (Invitrogen, Carlsbad, CA, USA). So that a mammalian G418 resistance cassette could be used for selection of targeting events, this plasmid was modified to remove the endogenous AphII gene (provides G418 resistance). The pKW4 contains LoxP (locus of X-ing over) sites that flank a G418 resistance cassette (based on mammalian codon usage), which is driven by a phosphoglycerate kinase (PGK) promoter (Lorson et al. 2011). This cassette was inserted into the RAG-1 gene to create the targeting construct pAB6. For RAG2, a 9466-bp PCR product i ncluding the RAG-2 gene was amplified and cloned into TOPO pCR-Blunt II (Invitrogen). The LoxP flanked G418 resistance cassette from pKW4 was inserted into the second exon of the RAG-2 gene sequence, creating the targeting construct pAB13. Further, diagnostic screening strategies were developed and validated to discriminate gene-targeting events from random integration. We report here 2 targeting vectors and validated screening methods for gene targeting in porcine fetal fibroblasts that have been validated for cloning. These vectors will be applied toward an effort to create a porcine SCID model. The implications of such a model include evaluation of basic immune function, evaluation of the innate immune system in vaccine efficacy, and organ transplantation.

Gene Targeting in the Red Alga Cyanidioschyzon merolae: Single- and Multi-Copy Insertion Using Authentic and Chimeric Selection Markers

The unicellular red alga Cyanidioschyzon merolae is an emerging model organism for studying organelle division and inheritance: the cell is composed of an extremely simple set of organelles (one nucleus, one mitochondrion and one chloroplast), and their genomes are completely sequenced. Although a fruitful set of cytological and biochemical methods have now been developed, gene targeting techniques remain to be fully established in this organism. Thus far, only a single selection marker, URACm-Gs, has been available that complements the uracil-auxotrophic mutant M4. URACm-Gs, a chimeric URA5.3 gene of C. merolae and the related alga Galdieria sulphuraria, was originally designed to avoid gene conversion of the mutated URA5.3 allele in the parental strain M4. Although an early example of targeted gene disruption by homologous recombination was reported using this marker, the genome structure of the resultant transformants had never been fully characterized. In the current study, we showed that the use of the chimeric URACm-Gs selection marker caused multicopy insertion at high frequencies, accompanied by undesired recombination events at the targeted loci. The copy number of the inserted fragments was variable among the transformants, resulting in high yet uneven levels of transgene expression. In striking contrast, when the authentic URA5.3 gene (URACm-Cm) was used as a selection marker, efficient single-copy insertion was observed at the targeted locus. Thus, we have successfully established a highly reliable and reproducible method for gene targeting in C. merolae. Our method will be applicable to a number of genetic manipulations in this organism, including targeted gene disruption, replacement and tagging.

A Targetron System for Gene Targeting in Thermophiles and Its Application in Clostridium thermocellum

BackgroundTargetrons are gene targeting vectors derived from mobile group II introns. They consist of an autocatalytic intron RNA (a “ribozyme”) and an intron-encoded reverse transcriptase, which use their combined activities to achieve highly efficient site-specific DNA integration with readily programmable DNA target specificity.Methodology/Principal FindingsHere, we used a mobile group II intron from the thermophilic cyanobacterium Thermosynechococcus elongatus to construct a thermotargetron for gene targeting in thermophiles. After determining its DNA targeting rules by intron mobility assays in Escherichia coli at elevated temperatures, we used this thermotargetron in Clostridium thermocellum, a thermophile employed in biofuels production, to disrupt six different chromosomal genes (cipA, hfat, hyd, ldh, pta, and pyrF). High integration efficiencies (67–100% without selection) were achieved, enabling detection of disruptants by colony PCR screening of a small number of transformants. Because the thermotargetron functions at high temperatures that promote DNA melting, it can recognize DNA target sequences almost entirely by base pairing of the intron RNA with less contribution from the intron-encoded protein than for mesophilic targetrons. This feature increases the number of potential targetron-insertion sites, while only moderately decreasing DNA target specificity. Phenotypic analysis showed that thermotargetron disruption of the genes encoding lactate dehydrogenase (ldh; Clo1313_1160) and phosphotransacetylase (pta; Clo1313_1185) increased ethanol production in C. thermocellum by decreasing carbon flux toward lactate and acetate.Conclusions/SignificanceThermotargetron provides a new, rapid method for gene targeting and genetic engineering of C. thermocellum, an industrially important microbe, and should be readily adaptable for gene targeting in other thermophiles.

Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha L.

The liverwort Marchantia polymorpha is an emerging model organism on account of its ideal characteristics for molecular genetics in addition to occupying a crucial position in the evolution of land plants. Here we describe a method for gene targeting by applying a positive/negative selection system for reduction of non-homologous random integration to an efficient Agrobacterium-mediated transformation system using M. polymorpha sporelings. The targeting efficiency was evaluated by knocking out the NOP1 gene, which impaired air-chamber formation. Homologous recombination was observed in about 2% of the thalli that passed the positive/negative selection. With the advantage of utilizing the haploid gametophytic generation, this strategy should facilitate further molecular genetic analysis of M. polymorpha, in which many of the mechanisms found in land plants are conserved, yet in a less complex form.

Targeted Gene Replacement in Drosophila Goes the Distance

> In this commentary, K. Nicole Crown and Jeff Sekelsky examine new methods for gene targeting in Drosophila . These methods, which allow for rapid replacement of large regions adjacent to existing transgenic insertions, are described in two articles published in this month's GENETICS: [“Long-

A General Approach for Controlling Transcription and Probing Epigenetic Mechanisms: Application to the Cd4 Locus

Synthetic regulatory proteins such as tetracycline (tet)-controlled transcription factors are potentially useful for repression as well as ectopic activation of endogenous genes and also for probing their regulatory mechanisms, which would offer a versatile genetic tool advantageous over conventional gene targeting methods. In this study, we provide evidence supporting this concept using Cd4 as a model. CD4 is expressed in double-positive and CD4 cells but irreversibly silenced in CD8 cells. The silencing is mediated by heterochromatin established during CD8 lineage development via transient action of the Cd4 silencer; once established, the heterochromatin becomes self-perpetuating independently of the Cd4 silencer. Using a tet-sensitive Cd4 allele harboring a removable Cd4 silencer, we found that a tet-controlled repressor recapitulated the phenotype of Cd4-deficient mice, inhibited Cd4 expression in a reversible and dose-dependent manner, and could surprisingly replace the Cd4 silencer to induce irreversible Cd4 silencing in CD8 cells, thus suggesting the Cd4 silencer is not the (only) determinant of heterochromatin formation. In contrast, a tet-controlled activator reversibly disrupted Cd4 silencing in CD8 cells. The Cd4 silencer impeded this disruption but was not essential for its reversal, which revealed a continuous role of the silencer in mature CD8 cells while exposing a remarkable intrinsic self-regenerative ability of heterochromatin after forced disruption. These data demonstrate an effective approach for gene manipulation and provide insights into the epigenetic Cd4 regulatory mechanisms that are otherwise difficult to obtain.

LoxP-FRT Trap (LOFT): a simple and flexible system for conventional and reversible gene targeting

BackgroundConditional gene knockout (cKO) mediated by the Cre/LoxP system is indispensable for exploring gene functions in mice. However, a major limitation of this method is that gene KO is not reversible. A number of methods have been developed to overcome this, but each method has its own limitations.ResultsWe describe a simple method we have named LOFT [LoxP-flippase (FLP) recognition target (FRT) Trap], which is capable of reversible cKO and free of the limitations associated with existing techniques. This method involves two alleles of a target gene: a standard floxed allele, and a multi-functional allele bearing an FRT-flanked gene-trap cassette, which inactivates the target gene while reporting its expression with green fluorescent protein (GFP); the trapped allele is thus a null and GFP reporter by default, but is convertible into a wild-type allele. The floxed and trapped alleles can typically be generated using a single construct bearing a gene-trap cassette doubly flanked by LoxP and FRT sites, and can be used independently to achieve conditional and constitutive gene KO, respectively. More importantly, in mice bearing both alleles and also expressing the Cre and FLP recombinases, sequential function of the two enzymes should lead to deletion of the target gene, followed by restoration of its expression, thus achieving reversible cKO. LOFT should be generally applicable to mouse genes, including the growing numbers of genes already floxed; in the latter case, only the trapped alleles need to be generated to confer reversibility to the pre-existing cKO models. LOFT has other applications, including the creation and reversal of hypomorphic mutations. In this study we proved the principle of LOFT in the context of T-cell development, at a hypomorphic allele of Baf57/Smarce1 encoding a subunit of the chromatin-remodeling Brg/Brahma-associated factor (BAF) complex. Interestingly, the FLP used in the current work caused efficient reversal in peripheral T cells but not thymocytes, which is advantageous for studying developmental epigenetic programming of T-cell functions, a fundamental issue in immunology.ConclusionsLOFT combines well-established basic genetic methods into a simple and reliable method for reversible gene targeting, with the flexibility of achieving traditional constitutive and conditional KO.

Reverse Genetic Approaches in Zebrafish

Zebrafish (Danio rerio) is a well-established vertebrate animal model. A comprehensive collection of reverse genetics tools has been developed for studying gene function in this useful organism. Morpholino is the most widely used reagent to knock down target gene expression post-transcriptionally. For a long time, targeted genome modification has been heavily relied on large-scale traditional forward genetic screens, such as ENU (N-ethyl-N-nitrosourea) mutagenesis derived TILLING (Targeting Induced Local Lesions IN Genomes) strategy and pseudo-typed retrovirus mediated insertional mutagenesis. Recently, engineered endonucleases, including ZFNs (zinc finger nucleases) and TALENs (transcription activator-like effector nucleases), provide new and efficient strategies to directly generate site-specific indel mutations by inducing double strand breaks in target genes. Here we summarize the major reverse genetic approaches for loss-of-function studies used and emerging in zebrafish, including strategies based on genome-wide mutagenesis and methods for site-specific gene targeting. Future directions and expectations will also be discussed.

Genome-wide identification of endothelial cell–enriched genes in the mouse embryo

AbstractThe early blood vessels of the embryo and yolk sac in mammals develop by aggregation of de novo–forming angioblasts into a primitive vascular plexus, which then undergoes a complex remodeling process. Angiogenesis is also important for disease progression in the adult. However, the precise molecular mechanism of vascular development remains unclear. It is therefore of great interest to determine which genes are specifically expressed in developing endothelial cells (ECs). Here, we used Flk1-deficient mouse embryos, which lack ECs, to perform a genome-wide survey for genes related to vascular development. We identified 184 genes that are highly enriched in developing ECs. The human orthologs of most of these genes were also expressed in HUVECs, and small interfering RNA knockdown experiments on 22 human orthologs showed that 6 of these genes play a role in tube formation by HUVECs. In addition, we created Arhgef15 knockout and RhoJ knockout mice by a gene-targeting method and found that Arhgef15 and RhoJ were important for neonatal retinal vascularization. Thus, the genes identified in our survey show high expression in ECs; further analysis of these genes should facilitate our understanding of the molecular mechanisms of vascular development in the mouse.

CELL BIOLOGY SYMPOSIUM: Novel technologies and novel insights1

The Cell Biology Symposium titled “Novel technologies and novel insights” was held at the Joint Annual Meeting of the American Society of Animal Science and the American Dairy Science Association in New Orleans, Louisiana, July 10 to 14, 2011. The purpose of the symposium was to provide the animal science community with an update on novel molecular technologies that allow new insights into genome structure and function in livestock. The first speaker of the symposium, Jeffrey Whyte (University of Missouri, Columbia), provided an overview of conventional and emerging technologies to modify the genome in mammals with specific examples in swine (Whyte and Prather, 2011). Engineered zinc finger nucleases (ZFN) have recently been developed that bind and cleave DNA at specific sequences and lead to targeting efficiencies up to 10,000-fold greater than conventional gene targeting methods with somatic cell nuclear transfer. The ZFN technology provides a precise mechanism to either disrupt a gene coding sequence (i.e., gene knockout) or enhance insertion of a DNA construct (i.e., transgenesis or gene knock-in). Several laboratories have now used ZFN technology to knockout genes in the swine genome. Availability of the draft swine genome sequence in combination with ZFN technology to modify the swine genome should allow more rapid and efficient development of swine models for the dual benefit of agricultural and biomedical research. Christian Haudenschild (Illumina, Hayward, CA) provided an overview of next-generation sequencing technologies and the novel use of these technologies to measure gene expression in various biological systems (Haudenschild, 2011). These technologies allow scientists to study temporal expression of the entire transcriptome in specific cells or tissues. In addition, nextgeneration sequencing technologies also reveal new insights regarding the transcription of specific alleles, splice variants, and a better definition of gene structure and annotation. Use of next-generation sequencing technologies to study gene expression has generated extremely large sets of data that require development of novel analytical and statistical methods for appropriate analysis and interpretation. The final speaker, Rebecca Wattam (Virginia Polytechnic Institute and State University, Blacksburg), described the opportunities and challenges associated with accessing all of the available public information to analyze and interpret large sets of genomic data (Sobral, 2011). Novel bioinformatic tools were developed to mine large data resources of genomic information. The examples of Brucella spp. and the 40 sequenced genomes of Brucella were used to show how genome sequencing could be used to identify common genes (and gene families) across species of Brucella, as well as missing areas of genome sequence in some species of Brucella. Specific genes and gene families that are associated with various physiological events, including preferences of the various species of Brucella for specific hosts, were identified. In addition, some species of Brucella are missing many large areas of genomic sequences that may be involved with virulence or synthesis of lipopolysaccharide. The recent availability of draft genome sequences for livestock coupled with emerging computational technologies developed by biomedical scientists provide the animal science community with an increasing number of novel tools to gain new insights into the structure and function of livestock genomes. The ultimate goal of these studies is to improve the efficiency of food and fiber production. The Cell Biology Symposium provided the animal science community with an update on novel molecular technologies to better understand the structure and function of livestock genomes. Use of ZFN technology provides opportunities to precisely

Development of an Efficient Agrobacterium-Mediated Gene Targeting System for Rice and Analysis of Rice Knockouts Lacking Granule-Bound Starch Synthase (Waxy) and β1,2-Xylosyltransferase

We have developed a high-frequency method for Agrobacterium-mediated gene targeting by combining an efficient transformation system using rice suspension-cultured calli and a positive/negative selection system. Compared with the conventional transformation system using calli on solid medium, transformation using suspension-cultured calli resulted in a 5- to 10-fold increase in the number of resistant calli per weight of starting material after positive/negative selection. Homologous recombination occurred in about 1.5% of the positive/negative selected calli. To evaluate the efficacy of our method, we show in this report that knockout rice plants containing either a disrupted Waxy (granule-bound starch synthase) or a disrupted Xyl (β1,2-xylosyltransferase) gene can be easily obtained by homologous recombination. Study of gene function using homologous recombination in higher plants can now be considered routine work as a direct result of this technical advance.

Efficient and Accurate Homologous Recombination in hESCs and hiPSCs Using Helper-dependent Adenoviral Vectors

Low efficiencies of gene targeting via homologous recombination (HR) have limited basic research and applications using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Here, we show highly and equally efficient gene knockout and knock-in at both transcriptionally active (HPRT1, KU80, LIG1, LIG3) and inactive (HB9) loci in these cells using high-capacity helper-dependent adenoviral vectors (HDAdVs). Without the necessity of introducing artificial DNA double-strand breaks, 7-81% of drug-resistant colonies were gene-targeted by accurate HR, which were not accompanied with additional ectopic integrations. Even at the motor neuron-specific HB9 locus, the enhanced green fluorescent protein (EGFP) gene was accurately knocked in in 23-57% of drug-resistant colonies. In these clones, induced differentiation into the HB9-positive motor neuron correlated with EGFP expression. Furthermore, HDAdV infection had no detectable adverse effects on the undifferentiated state and pluripotency of hESCs and hiPSCs. These results suggest that HDAdV is one of the best methods for efficient and accurate gene targeting in hESCs and hiPSCs and might be especially useful for therapeutic applications.

Crosstalk Between Cyclins D1 and D3 in Mantle Cell Level At the Transcriptional and Postranscriptional Levels

Abstract 1373Cyclin D1 (CCND1) expression is deregulated epigenetically in mantle cell lymphoma by the t(11;14) chromosome translocation. We have investigated the phenomenon of cyclin D1 mediated oncogene addiction in mantle cell lymphoma (MCL) and multiple myeloma (MM) cell lines. Gene targeting methods were utilized to generate Cyclin D1(-) MCL and MM cell lines. These cell lines did not make any detectable cyclin D1 mRNA and protein. These cells lines had shorter doubling times in vitro than their cyclin D1(+) counterparts and were also more chemoresistant in vitro and more tumorigenic in immunodeficient mice. Cyclin D3 mRNA and protein levels were increased dramatically. Q-RTPCR analysis showed a 17 fold increase in the mRNA of cyclin D3. The half-life of cyclin D3 protein was also significantly prolonged, from 20 minutes in wild type cells to 5 hours in mutant cyclin. Knockdown of cyclin D3 dramatically inhibited cell growth and caused apoptosis in the cyclin D1(-) but not in the t(11;14) cyclin D1 positive parental cells. Compensation and crosstalk between the cyclin D1 and D3 genes in MCL and MM cells were shown both in vitro and in vivo. Thus, the addiction to cyclin D1 in MCL and MM cells can be substituted by cyclin D3. Therapies that can target both cyclins D1 and D3 posttranscriptionally such as curcumin in combination with bortezomib were investigated in vitro in MCL cells. Combination treatment of MCL and MM cell lines with these agents produce significant downregulation of the protein levels of cyclins D1 and D3, even in cyclin D1(-) cell lines containing a extremely stable cyclin D3 protein.In addition, we have shown that iron chelators such as desferoxamine can turn off cyclin D1 transcription and cause apoptosis of MCL cells in vitro.Published work from several laboratories has described cyclin dependent kinase (Cdk) independent DNA binding activity for cyclin D1. Evidence that cyclin D1 and D3 might be involved in the pathogenesis of MCL in a cdk independent manner was observed.We confirmed that cyclin D1 is a DNA binding protein and demonstrated by chromatin immuprecipitation assays that it binds to the endogenous cyclin D3 gene promoter both in a MCL cell line and in cells from leukemic MCL patients. The binding site corresponds to a published E2F binding site upstream of exon 2, which regulates two cyclin D3 transcript variants. We have also identified several potential targets of cyclin D1 regulation based on published cyclin D1 microarray experiments, several of which exhibit promoter- enrichment following cyclin D1 CHIP: GSK3b, ID3, and ANSN. Thus, cyclin D1 is a potential repressor of cyclin D3 expression by direct binding to the cyclin D3 promoter.These data demonstrate crosstalk between cyclin D1 and D3 at both the transcriptional and post transcriptional levels. Furthermore, we demonstrate for the first time that cyclin D1 binds to the cyclin D3 promoter and potentially can mediate repression of cyclin D3 mRNA transcription. Deletion of cyclin D1 in an MCL cell line results in significant upregulation cyclin D3 mRNA and protein levels and a dramatic increase in the half life of cyclin D3 in cyclin D1 null cells. Previously reported data using knockdown and antisense technologies in MCL cell lines would be unable to appreciate these observations because of residual cyclin D1 mRNA and protein levels. Agents such as bortezomib and curcumin that downregulate cyclin D1 protein levels and iron chelating agents that downregulate cyclin D1 RNA levels may be useful agents in combination treatment of MCL and MM. [Display omitted] Disclosures:Epner:Merck: Consultancy, Honoraria, Speakers Bureau; Novartis: Speakers Bureau; Millenium: Speakers Bureau; Allos: Speakers Bureau; Enzon: Speakers Bureau; GSK: Speakers Bureau.

CELL BIOLOGY SYMPOSIUM: Zinc finger nucleases to create custom-designed modifications in the swine (Sus scrofa) genome1,2

Engineered zinc finger nucleases (ZFN) are rapidly gaining popularity as a means to enhance the rate and specificity of DNA modifications in plant and animal cells. Repair-mediated gene modification by ZFN is driven by introducing DNA double-strand breaks via a nonspecific nuclease domain linked to a sequence-specific zinc finger nucleotide recognition domain. This review examines the use of ZFN to produce genetically modified swine and the potential of this technology for the future. By combining conventional gene targeting methods with somatic cell nuclear transfer, several genetically modified pig models have been produced. These conventional techniques are inefficient in mammalian somatic cells and provide little control over the site specificity and rate of exogenous DNA integration. The use of engineered ZFN that bind and cleave genomic DNA at specific loci can enhance targeting efficiencies by orders of magnitude. Recent publication of the first genetic modification in pigs by combining ZFN technology with somatic cell nuclear transfer has opened the door to genome targeting with a precision that was not previously possible in a large animal model. Since that time, model pigs with selective knockout of endogenous genes have been produced. This review will examine the use of ZFN to generate these pig models and the potential of ZFN to accelerate the production of genetically modified pigs of agricultural and biomedical importance. Current methods of ZFN design, important considerations for their safe and effective use in modification of the swine genome, and future innovative applications of this technology in pigs will be discussed.

Reproducible gene targeting in recalcitrant Escherichia coli isolates

BackgroundA number of allele replacement methods can be used to mutate bacterial genes. For instance, the Red recombinase system of phage Lambda has been used very efficiently to inactivate chromosomal genes in E. coli K-12, through recombination between regions of homology. However, this method does not work reproducibly in some clinical E. coli isolates.FindingsThe procedure was modified by using longer homologous regions (85 bp and 500-600 bp), to inactivate genes in the uropathogenic E. coli strain UTI89. An lrhA regulator mutant, and deletions of the lac operon as well as the complete type 1 fimbrial gene cluster, were obtained reproducibly. The modified method is also functional in other recalcitrant E. coli, like the avian pathogenic E. coli strain APEC1. The lrhA regulator and lac operon deletion mutants of APEC1 were successfully constructed in the same way as the UTI89 mutants. In other avian pathogenic E. coli strains (APEC3E, APEC11A and APEC16A) it was very difficult or impossible to construct these mutants, with the original Red recombinase-based method, with a Red recombinase-based method using longer (85 bp) homologous regions or with our modified protocol, using 500 - 600 bp homologous regions.ConclusionsThe method using 500-600 bp homologous regions can be used reliably in some clinical isolates, to delete single genes or entire operons by homologous recombination. However, it does not invariably show a greater efficiency in obtaining mutants, when compared to the original Red-mediated gene targeting method or to the gene targeting method with 85 bp homologous regions. Therefore the length of the homology regions is not the only limiting factor for the construction of mutants in these recalcitrant strains.

Hybrid Capture and Next-Generation Sequencing Identify Viral Integration Sites from Formalin-Fixed, Paraffin-Embedded Tissue

Although next-generation sequencing (NGS) has been the domain of large genome centers, it is quickly becoming more accessible to general pathology laboratories. In addition to finding single-base changes, NGS allows for the detection of larger structural variants, including insertions/deletions, translocations, and viral insertions. We describe the use of targeted NGS on DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue, and show that the short read lengths of NGS are ideally suited to fragmented DNA obtained from FFPE tissue. Further, we describe a novel method for performing hybrid-capture target enrichment using PCR-generated capture probes. As a model, we captured the 5.3-kb Merkel cell polyomavirus (MCPyV) genome in FFPE cases of Merkel cell carcinoma using inexpensive, PCR-derived capture probes, and achieved up to 37,000-fold coverage of the MCPyV genome without prior virus-specific PCR amplification. This depth of coverage made it possible to reproducibly detect viral genome deletions and insertion sites anywhere within the human genome. Out of four cases sequenced, we identified the 5' insertion sites in four of four cases and the 3' sites in three of four cases. These findings demonstrate the potential for an inexpensive gene targeting and NGS method that can be easily adapted for use with FFPE tissue to identify large structural rearrangements, opening up the possibility for further discovery from archival tissue.

AAV-mediated gene targeting methods for human cells.

Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10(-5) to 10(-2) per infected cell. These targeting frequencies are 1-4 logs higher than those obtained by conventional transfection or electroporation approaches. A wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. Optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by Southern blots. This protocol (from vector design through a single round of targeting and screening) can be completed in ∼10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks.

Investigating the genetics of visual processing, function and behaviour in zebrafish

Over the past three decades, the zebrafish has been proven to be an excellent model to investigate the genetic control of vertebrate embryonic development, and it is now also increasingly used to study behaviour and adult physiology. Moreover, mutagenesis approaches have resulted in large collections of mutants with phenotypes that resemble human pathologies, suggesting that these lines can be used to model diseases and screen drug candidates. With the recent development of new methods for gene targeting and manipulating or monitoring gene expression, the range of genetic modifications now possible in zebrafish is increasing rapidly. Combined with the classical strengths of the zebrafish as a model organism, these advances are set to substantially expand the type of biological questions that can be addressed in this species. In this review, we outline how the potential of the zebrafish can be harvested in the context of eye development and visual function. We review recent technological advances used to study the formation of the eyes and visual areas of the brain, visual processing on the cellular, subcellular and molecular level, and the genetics of visual behaviour in vertebrates.