Targeted Gene Integration Research Articles

Targeted gene integration using the combination of a sequence-specific DNA-binding protein and phiC31 integrase

PhiC31 integrase-based vectors can integrate therapeutic genes selectively into attP or pseudo-attP sites in genomes, but considerable numbers of pseudo-attP sites in human genomes exist inside endogenous gene-coding regions. To avoid endogenous gene disruptions, we aimed to enhance the integration site-specificity of the phiC31 integrase-based vector using a sequence-specific DNA-binding protein containing Gal4 and LexA DNA-binding motifs. The dual DNA-binding protein was designed to tether the UAS-containing donor vector to the target sequence, the LexA operator, and restrict integration to sites close to the LexA operator. To analyze the site-specificity in chromosomal integration, a human cell line having LexA operators on the genome was established, and the cell line was transfected with donor vectors expressing the DNA-binding protein and the phiC31 integrase expression vector (helper vector). Quantitative PCR indicated that integration around the LexA operator was 26-fold higher with the UAS-containing donor vector than with the control. Sequence analysis confirmed that the integration occurred around the LexA operator. The dual DNA-binding protein-based targeted integration strategy developed herein would allow safer and more reliable genetic manipulations for various applications, including gene and cell therapies.

Cre-loxP-based system for removal and reuse of selection markers in Ashbya gossypii targeted engineering

The filamentous ascomycete Ashbya gossypii is amenable to genetic manipulation and is an excellent model system for studying eukaryotic cell biology. However, the number of selection markers in current use for both targeted gene integration and disruption in this fungus are very limited. Therefore, the Cre-loxP recombination system was adapted for use in A. gossypii and its effectiveness in recycling marker genes was demonstrated by constructing both single and double deleted Agura3 and Agade1 auxotrophic strains free of exogenous markers. In spite of its wide use, this is the first report in which the Cre-loxP system was applied to A. gossypii, opening new perspectives for targeted engineering of this fungus with several promising biotechnological applications.

Molecular tools and protocols for engineering the acid-tolerant yeast Zygosaccharomyces bailii as a potential cell factory.

Microorganisms offer a tremendous potential as cell factories, and they are indeed used by humans for centuries for biotransformations. Among them, yeasts combine the advantage of unicellular state with a eukaryotic organization, and, in the era of biorefineries, their biodiversity can offer solutions to specific process constraints. Zygosaccharomyces bailii, an ascomycetales budding yeast, is widely known for its peculiar tolerance to various stresses, among which are organic acids. Despite the possibility to apply with this yeast some of the molecular tools and protocols routinely used to manipulate Saccharomyces cerevisiae, adjustments and optimizations are necessary. Here, we describe in detail protocols for transformation, for target gene disruption or gene integration, and for designing episomal expression plasmids helpful for developing and further studying the yeast Z. bailii.

Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.

An efficient strategy for TALEN-mediated genome engineering in Drosophila

In reverse genetics, a gene’s function is elucidated through targeted modifications in the coding region or associated DNA cis-regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila. We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F1 generation.

Solution Scattering Studies of Large Serine Recombinase-DNA Complexes

Large serine recombinase (LSR) proteins are emerging as desirable tools in targeted gene therapy due to their ability to catalyze the integration of any target DNA into a host genome in a highly specific and unidirectional manner. They have long been shown to mediate the integration and long-term expression of target genes in human stem cell and lung and liver cancer cell lines. LSRs consist of a catalytic domain, containing the active site serine, and a large C-terminal domain responsible for DNA binding. The LSR protein recognizes unique attachment sites in the target and host DNA which confer the high specificity of LSR mediated recombination. Based on current models, recombination is completed when two subunits of a tetrameric recombinase complex undergo a 180° rotation, generating new attachment sites that discourage excision of the target DNA. However, there are currently no published structures of a LSR protein alone or bound to DNA to understand the mechanism of this exquisite directionality. We are close to completing the first crystal structure of a LSR bound to DNA which will provide insight into the high specificity of the LSR-DNA interaction. In addition, we have employed small-angle x-ray scattering (SAXS) and small-angle neutron scattering (SANS) techniques to determine low resolution structures of the LSR-DNA complex intermediates from the integration pathway. Detailed structural models of these molecular intermediates are essential in determining how this recombination reaction occurs.

Molecular Characterization of Transgene Integration by Next-Generation Sequencing in Transgenic Cattle

As the number of transgenic livestock increases, reliable detection and molecular characterization of transgene integration sites and copy number are crucial not only for interpreting the relationship between the integration site and the specific phenotype but also for commercial and economic demands. However, the ability of conventional PCR techniques to detect incomplete and multiple integration events is limited, making it technically challenging to characterize transgenes. Next-generation sequencing has enabled cost-effective, routine and widespread high-throughput genomic analysis. Here, we demonstrate the use of next-generation sequencing to extensively characterize cattle harboring a 150-kb human lactoferrin transgene that was initially analyzed by chromosome walking without success. Using this approach, the sites upstream and downstream of the target gene integration site in the host genome were identified at the single nucleotide level. The sequencing result was verified by event-specific PCR for the integration sites and FISH for the chromosomal location. Sequencing depth analysis revealed that multiple copies of the incomplete target gene and the vector backbone were present in the host genome. Upon integration, complex recombination was also observed between the target gene and the vector backbone. These findings indicate that next-generation sequencing is a reliable and accurate approach for the molecular characterization of the transgene sequence, integration sites and copy number in transgenic species.

Characterization of Rad51 from Apicomplexan Parasite Toxoplasma gondii: An Implication for Inefficient Gene Targeting

Repairing double strand breaks (DSBs) is absolutely essential for the survival of obligate intracellular parasite Toxoplasma gondii. Thus, DSB repair mechanisms could be excellent targets for chemotherapeutic interventions. Recent genetic and bioinformatics analyses confirm the presence of both homologous recombination (HR) as well as non homologous end joining (NHEJ) proteins in this lower eukaryote. In order to get mechanistic insights into the HR mediated DSB repair pathway in this parasite, we have characterized the key protein involved in homologous recombination, namely TgRad51, at the biochemical and genetic levels. We have purified recombinant TgRad51 protein to 99% homogeneity and have characterized it biochemically. The ATP hydrolysis activity of TgRad51 shows a higher KM and much lower kcat compared to bacterial RecA or Rad51 from other related protozoan parasites. Taking yeast as a surrogate model system we have shown that TgRad51 is less efficient in gene conversion mechanism. Further, we have found that TgRad51 mediated gene integration is more prone towards random genetic loci rather than targeted locus. We hypothesize that compromised ATPase activity of TgRad51 is responsible for inefficient gene targeting and poor gene conversion efficiency in this protozoan parasite. With increase in homologous flanking regions almost three fold increments in targeted gene integration is observed, which is similar to the trend found with ScRad51. Our findings not only help us in understanding the reason behind inefficient gene targeting in T. gondii but also could be exploited to facilitate high throughput knockout as well as epitope tagging of Toxoplasma genes.

Flipase‐mediated cassette exchange in Sf9 insect cells for stable gene expression

Site-specific DNA integration allows predictable heterologous gene expression and circumvents extensive clone screening. Herein, the establishment of a Flipase (Flp)-mediated cassette exchange system in Sf9 insect cells for targeted gene integration is described. A tagging cassette harboring a reporter dsRed gene was randomly introduced into the cell genome after screening different transfection protocols. Single-copy integration clones were then co-transfected with both Flp-containing plasmid and an EGFP-containing targeting cassette. Successful cassette exchange was suggested by emergence of G418-resistant green colonies and confirmed by PCR analysis, showing the absence of the tagging cassette and single integration of the targeting cassette in the same locus. Upon cassette exchange, uniform EGFP expression between clones derived from the same integration site was obtained. Moreover, the resulting cell clones exhibited the expression properties of the parental cell line. EGFP production titers over 40 mg/L were of the same order of magnitude as those achieved through baculovirus infection. This Sf9 master cell line constitutes a versatile and re-usable platform to produce multiple recombinant proteins for fundamental and applied research.

Gene Expression Profiling in Multiple Myeloma—Reporting of Entities, Risk, and Targets in Clinical Routine

Multiple myeloma is an incurable malignant plasma cell disease characterized by survival ranging from several months to more than 15 years. Assessment of risk and underlying molecular heterogeneity can be excellently done by gene expression profiling (GEP), but its way into clinical routine is hampered by the lack of an appropriate reporting tool and the integration with other prognostic factors into a single "meta" risk stratification. The GEP-report (GEP-R) was built as an open-source software developed in R for gene expression reporting in clinical practice using Affymetrix microarrays. GEP-R processes new samples by applying a documentation-by-value strategy to the raw data to be able to assign thresholds and grouping algorithms defined on a reference cohort of 262 patients with multiple myeloma. Furthermore, we integrated expression-based and conventional prognostic factors within one risk stratification (HM-metascore). The GEP-R comprises (i) quality control, (ii) sample identity control, (iii) biologic classification, (iv) risk stratification, and (v) assessment of target genes. The resulting HM-metascore is defined as the sum over the weighted factors gene expression-based risk-assessment (UAMS-, IFM-score), proliferation, International Staging System (ISS) stage, t(4;14), and expression of prognostic target genes (AURKA, IGF1R) for which clinical grade inhibitors exist. The HM-score delineates three significantly different groups of 13.1%, 72.1%, and 14.7% of patients with a 6-year survival rate of 89.3%, 60.6%, and 18.6%, respectively. GEP reporting allows prospective assessment of risk and target gene expression and integration of current prognostic factors in clinical routine, being customizable about novel parameters or other cancer entities.

Metascoring and Gene Expression Profiling in Clinical Routine in Multiple Myeloma,

Abstract 3940▪▪This icon denotes a clinically relevant abstract BACKGROUND.Multiple myeloma is characterized by molecular heterogeneity transmitting to survival ranging from several months to over 15 years. Gene expression profiling allows assessment of biological entities, risk, and targets. Its translation into clinical routine alongside conventional prognostic factors has been prevented by lack of appropriated reporting tools and the integration with other prognostic factors into a single risk stratification (metascoring). METHODS.We present here a non-commercial open source software-framework developed in the open source language R (GEP-report) containing a graphic user interphase based on Gtk2. Affymetrix microarray raw-data and a documentation-by-value strategy to directly apply thresholds and grouping-algorithms from a reference cohort of 262 myeloma patients are used. Gene expression-based and conventional prognostic factors are integrated within one risk-stratification (HM-metascore) and the final report is created as a PDF-file. RESULTS.The GEP-report comprises i) quality control, ii) test of sample identity, iii) biological classifications of multiple myeloma, iv) risk stratification, v) assessment of target-genes, and vi) integration of expression-based and clinical risk factors within one metascore. This HM-metascore sums over the weighted factors gene-expression based risk-assessment (UAMS-, IFM-score), proliferation, ISS-stage, t(4;14), and expression of prognostic target-genes (AURKA, IGF1R) for which clinical grade inhibitors exist. It delineates three significantly different groups of 13.1, 72.1 and 14.7% of patients with a 6-year survival of 89.3, 60.6 and 18.6%, respectively. CONCLUSION.GEP-reporting allows prospective assessment of target gene expression and integration of current prognostic factors within one risk stratification (metascoring), being customizable regarding novel parameters or other cancer entities. Disclosures:No relevant conflicts of interest to declare.

422. Targeted Gene Integration into a Mouse Safe Harbor Locus Directed by Engineered Zinc Finger Nucleases

422. Targeted Gene Integration into a Mouse Safe Harbor Locus Directed by Engineered Zinc Finger Nucleases

633. Targeted Gene Integration in Human Epidermal Stem Cells by Zinc-Finger Nuclease-Mediated Homologous Recombination

633. Targeted Gene Integration in Human Epidermal Stem Cells by Zinc-Finger Nuclease-Mediated Homologous Recombination

Regeneration of transgenic plants from cell suspensions of transformed apple (Malus domestica Borkh. cv. Gala)

SummaryShoot regeneration in most apple cultivars is sensitive to antibiotic selection, thus Agrobacterium-mediated transformation is of low efficiency. Our experiments confirmed that, with a moderate selection pressure (10 or 20 mg l–1 kanamycin), a high percentage of callus formation (67.0% or 54.8%, respectively) was observed. Over 80.0% or 95.6% of these calli were transgenic, although the efficiency of transgenic shoot regeneration was low. Modifying the kanamycin selection pressure, or shortening the time of kanamycin selection to 2 weeks, did not increase the efficiency of transformation. Multiplying suspension cultures could be produced from transgenic calli using single cells or small cell aggregates to initiate such cultures. When these transgenic cell aggregates were placed on regeneration medium without kanamycin selection, 17.3% of them regenerated shoots after 8 weeks, and at least 86 transgenic plants were obtained from one suspension culture line. This level of success makes it possible to obtain large numbers of transgenic apple plants from a single experiment for genetic breeding, and to study the mechanisms of integration of target genes.

An accumulative site‐specific gene integration system using cre recombinase‐mediated cassette exchange

The Cre-loxP system is frequently used for site-specific recombination in animal cells. The equilibrium and specificity of the recombination reaction can be controlled using mutated loxPs. In the present study, we designed an accumulative site-specific gene integration system using Cre recombinase and mutated loxPs in which the Cre-mediated cassette exchange reaction is infinitely repeatable for target gene integration into loxP target sites. To evaluate the feasibility and usefulness of this system, a series of integration reactions were repeated and confirmed in vitro using Cre recombinase protein and plasmids. Accumulative gene integration was also performed on the genome of Chinese hamster ovary (CHO) cells. The results indicated that the system was applicable for repeated gene integration of multiple genes to the target sites on both plasmids and CHO cell genomes. This gene integration system provides a novel strategy for gene amplification and for biological analyses of gene function through the genetic modification of cells and organisms.

Random and targeted gene integrations through the control of non‐homologous end joining in the yeast Kluyveromyces marxianus

Kluyveromyces marxianus DMKU3-1042 is a thermotolerant yeast strain suitable for high-temperature ethanol fermentation and genetic engineering with linear DNA. We have developed a highly efficient random gene integration method with a frequency that exceeds 2.5 x 10(6) transformants/microg linear DNA, a figure comparable to what is observed with autonomously replicating plasmid transformation in Saccharomyces cerevisiae. To establish the mechanism of random integration in DMKU3-1042, we identified and deleted the K. marxianus KU70 gene, which is known to be involved in the non-homologous end-joining (NHEJ) pathway. In yeast lacking KU70, high-frequency non-homologous gene integration was abolished and the Kmku70 mutants showed 82-95% homologous gene targeting efficiencies using homologous sequences of 40-1000 bp. These results indicate that the highly efficient NHEJ pathway can be utilized with random gene disruption techniques such as transposon mutagenesis and plasmid-free gene manipulations in K. marxianus.

Targeted gene modifications in drug discovery and development

In the present review, we discuss technologies and principles guiding the choice of cell-based assays. We show that a major trend is to expedite cell line development in cellular systems [stem cells, induced pluripotent stem cells (iPS), primary cells ...] that may reflect the physiology and differentiation of cells in living organs. We discuss different expression technologies and propose that targeted gene integration is the best approach, applicable to all types of cells. We propose that targeted gene expression combined with the development of assays that approach the physiology of the human body, by creating better screening tools, may help to reduce failure and attrition rate at late stages of clinical development.

Agrobacterium-mediated co-transformation of multiple genes in upland cotton

Two cotton genotypes, Simian 3 (SM 3) and WC, were co-transformed using a mixture of four Agrobacterium tumefaciens cultures of strain LBA4404, each carrying a plasmid harboring the following genes, Bt + sck (for Bacillus thuringenesis protein and modified Cowpea trypsin inhibitor), bar (for glufosinate), keratin, and fibroin. The frequency of callus induction, embryogenesis, and plant regeneration were notably different between the two genotypes. However, there were no differences between the two genotypes for number of plantlets carrying multiple gene copies of different gene combinations as well as transformation frequency for different gene combinations. PCR analysis indicated that more than 80% of plantlets carried the nptII gene for kanamycin resistance. Overall, the co-transformation frequency of two or more genes was about 35%. Southern blot analysis confirmed integration of target genes into the cotton genome, and the number of copies of the transgene(s) varied from one to four. Multiple transgene expression was confirmed by RT-PCR analysis in some transgenic lines. Further analysis of T1 plants demonstrated that multiple transgenes were inherited and expressed in progenies.

두 가지 제초제에 대하여 저항성을 가지는 항생제 마커-프리 형질전환 감자 육성

본 연구에서는 제초제 저항성 bar 유전자 및 CP4-EPSPS 유전자를 포함하는 발현벡터로 형질전환되고 항생제 마커 유전자를 포함하지 않는 제초제 복합 저항성 감자 식물체를 육성하고자 실험하였다. Bar 유전자를 포함하는 pCAMBIA3300에 CaMV35S 프로모터에 의해 조절되는 CP4-EPSPS 유전자를 도입하여 식물체용 발현 운반체를 제작하고, 이를 Agrobacterium tumafaciens EHA105에 도입하였다. 태동밸리 잎 절편체를 Agrobacterium과 공동배양한 다음, phosphinothricin 0.5 mg/L이 첨가된 배지에서 선발하고 호르몬 무처리 MS발근시켜 형질전환체 (E3-6)를 얻었다. PCR, Southern 분석, 효소면역반응 분석 등을 통해 두 가지 유전자가 도입되었으며 이들이 정상적으로 발현됨이 확인되었다. E3-6 식물체는 glufosinate-ammonium의 어린 식물체 잎 도포처리, glyphosate 용액에 치상한 식물체 조직에서의 shikimate 축적 여부 조사를 통하여 조사한 결과, 두 제초제에 대해 저항성을 나타내었다. 또한 형질전환감자의 전식물체에 대해 glyphosate와 glufosinate-ammonium 각각의 용액 또는 이들의 혼합물을 처리한 후 제초활성 반응을 조사한 결과, E3-6 형질전환 감자는 두 제초제를 각각 단독으로 처리할 때나 혼합하여 동시 처리할 때에도 동일한 저항성이 나타남을 확인하였다. This study was conducted to develop an antibiotics marker-free potato (Solanum tuberosum L., cv. Taedong valley) plant having resistance against two herbicides. Agrobacterium tumefaciens strain EHA105, harboring a binary vector plasmid pCAMBIA3300 containing bar gene under the control of a promoter CaMV35S and linked CP4-EPSPS genes driven by CaMV35S promoter, was used in the current study. The leaf segments of newly bred potato variety (cv. Taedong Valley) was co-cultured with Agrobacterium. Then, the regenerated individual shoots were excised and transferred to potato multiplication medium supplemented with 0.5 mg/L phosphinothricin. The shoots were rooted in MS medium without hormone and obtained putative transgenic plant E3-6. Integration of target genes into the E3-6 plant and their expression was confirmed by PCR, Southern analysis, and ELISA test. The tissue necrosis test on young leaf blade and shikimic acid accumulation test using the tissue of E3-6 plant were conducted to investigate the resistance to glufosinate-ammonium and glyphosate, respectively. The transgenic plants (E3-6) simultaneously showed a high resistance to both herbicides. The same results were surely obtained also in the whole plants foliar-treated with alone or mixture of two herbicides, glufosinate-ammonium and glyphosate.

Six new amino acid-auxotrophic markers for targeted gene integration and disruption in fission yeast

Fission yeast Schizosaccharomyces pombe is amenable to genetics and is an excellent model system for studying eukaryotic cell biology. However, auxotrophic markers that can be used for both targeted gene integration and disruption are very limited. Here we performed a forward genetic screen in an effort to develop a new set of selectable markers for use in this yeast. Mutants that were auxotrophic for arginine, asparagine, cysteine, lysine, methionine and phenylalanine were isolated. Six genes were analyzed in detail and the mutations in the genes were identified. Among these six are three new genes: asn1 (+), cys2 (+) and pha2 (+) were required for biosynthesis of asparagine, cysteine and phenylalanine, respectively. New alleles of arg1 (+), lys3 (+) and met6 (+) were also identified. All of these genes proved to be suitable as selectable markers for targeted gene integration and disruption. We also showed that in Schizosaccharomyces pombe there are two apparent homologues of Saccharomyces cerevisiae MET2: the previously known met6 (+), and SPBC106.17c (named cys2 (+)). The cys2 mutation required cysteine rather than methionine. These new tools, specifically, new selectable markers, will be useful in further genetic and biological studies in fission yeast.