One-step generation of recombineering constructs by asymmetric-end ligation and negative selection

Ohtsuka, Masato; Mizutani, Akiko; Kikuti, Yukie Y.; Kulski, Jerzy K.; Kimura, Minoru; Tanaka, Masafumi; Inoko, Hidetoshi

doi:10.1016/j.ab.2006.05.001

Cited by 6 publications

(9 citation statements)

References 7 publications

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“…The targeting construct for H2-Tw3 gene (19) was generated by modifying the citb585c7 BAC clone (from a CITB library derived from the 129/SV mouse strain, Research Genetics/Invitrogen, Carlsbad, CA, USA) using recombineering method with the DY380 Escherichia coli (20) and the plasmids, pADY and pAEF (21). The protocols for recombineering fragments, of which homology regions (HRs) were PCR-amplified from the BAC clone with the following primer sets: (M393 and M394 for HR1, M395 and M396 for HR2, M397 and M398 for HR3 and M399 and M155 for HR4; Supplementary Table S1), were generated based on the protocols described previously by our group (21).…”

Section: Methodsmentioning

confidence: 99%

“…The protocols for recombineering fragments, of which homology regions (HRs) were PCR-amplified from the BAC clone with the following primer sets: (M393 and M394 for HR1, M395 and M396 for HR2, M397 and M398 for HR3 and M399 and M155 for HR4; Supplementary Table S1), were generated based on the protocols described previously by our group (21). The recombineering protocol was based on the protocol of Liu et al .…”

Section: Methodsmentioning

confidence: 99%

“…HRs for recombineering fragments were PCR-amplified from the BAC clone with the following primer sets: M058 and M057 for HR1, M056 and M055 for HR2, M060 and M059 for HR3 and M054 and M053 for HR4 (Supplementary Table S1). Recombineering fragments were prepared according to the method described previously by our group (21), and used for targeting vector construction. The resulting targeting vector (pAIW) had the following components in a 5′ to 3′ direction: DT-A cassette, short arm, SA, FRT sequence-fusion neo , JT15, IRES, ECFP-Nuc, polyA site, lox2272, long arm and I- Sce I site.…”

Section: Methodsmentioning

confidence: 99%

“…As for the promoterless reporter gene, lacZ gene was used for pAJK, pAMF, pAKB, pAMG, pAMJ, pANQ, pAOB, pAOF, pAOK, pAOL, pAOM, pAOT and pAOU, EGFP gene was used for pA748 and EYFP-Nuc gene was used for pA617. In the DOI region, the following cassettes were included in each donor vector: ‘dual SacB genes with GFPuv ’ (21) in pAJK and pA748, ‘CMV-HcRed1-polyA’ in pA617, ‘CAG-EGFP-polyA’ in pAMF, ‘FRT-fusion EGFP-polyA’ in pAKB, ‘CAG-EGFP-miR(Hr-1)-polyA’ in pAMG, ‘CAG-EGFP-miR(Hr-2)-polyA’ in pAMJ, ‘CAG-tau/kikume-polyA’ in pANQ, ‘CAG-mCherry-polyA’ in pAOB, ‘CAG-mCitrine-polyA’ in pAOF, ‘CAG-mCFP-polyA’ in pAOK, ‘CAG-mOrange-polyA’ in pAOL, ‘CAG-tdTomato-polyA’ in pAOM, ‘CAG-EGFP-miR(Tyr-1/2)-polyA’ in pAOT and ‘CAG-EGFP-miR(Tyr-3/4)-polyA’ in pAOU.…”

Section: Methodsmentioning

confidence: 99%

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Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression

Ohtsuka¹,

Ogiwara²,

Miura³

et al. 2010

Nucleic Acids Research

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108

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Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo ‘gain-of-function’ and ‘loss-of-function’ studies.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression

Ohtsuka¹,

Ogiwara²,

Miura³

et al. 2010

Nucleic Acids Research

Self Cite

108

View full text Add to dashboard Cite

show abstract

“…Some of these methods have been used to construct gene targeting constructs for various trypanosomatids [10,11]. However, these techniques can involve tedious, complicated, or unfamiliar technology [5,6,8,9,12], require expensive kits or reagents [4,8], and are complicated by inefficiency [13]. …”

mentioning

confidence: 99%

A rapid, efficient and economical method for generating leishmanial gene targeting constructs

Fulwiler

Soysa

Ullman

et al. 2011

Molecular and Biochemical Parasitology

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Targeted gene replacement is a powerful tool in Leishmania genetics that can be time-consuming to implement. One tedious aspect that delays progress is the multi-step construction of gene targeting vectors. To accelerate this process, we developed a streamlined method that allows the assembly of a complete targeting vector from all its constituent parts in a single-step multifragment ligation. The individual components to be assembled are flanked by sites for the restriction endonuclease SfiI that generates nonidentical, non-palindromic three base 3'-overhangs designed to allow annealing and ligation of the parts only in the proper order. The method was optimized by generating constructs for targeting the Leishmania donovani inosine monophosphate dehydrogenase gene (LdIMPDH) encoding six different drug resistance markers, and was found to be rapid and efficient. These constructs were successfully employed to generate heterozygous LdIMPDH gene replacement mutants. This method is adaptable for generating targeting vectors for a variety of species. KeywordsLeishmania; gene targeting; gene knockout; homologous recombination; multi-fragment ligation; SfiI restriction endonuclease Targeted gene replacement via homologous recombination has been an invaluable tool for the genetic dissection of important metabolic and virulence pathways in Leishmania species [1], as well as for many other protozoan parasites [2]. The general experimental approach for the genetic manipulation of model organisms is essentially the same: DNA sequences of sufficient length to direct homologous recombination flanking the gene to be targetedreferred to herein as 5'-and 3'-targeting sequences (TS) -are independently isolated and joined to an alternative gene (i.e., drug resistance gene) that allows selection of cells in which the appropriate integration event has occurred. The most common method for generating leishmanial and other parasite gene targeting constructs involves the sequential cloning of 5'-TS and 3'-TS DNAs into a vector encoding a drug resistance cassette flanked by restriction sites [3]. This multi-step process can be time consuming and is complicated by the fact that commonly used vectors have limited restriction sites for TS insertion, exchange of drug resistance markers, and excision of the targeting cassette from the vector backbone * Corresponding Author. 3181 SW Sam Jackson Park Road, 7320 Richard T. Jones Hall, Portland,; yatesp@ohsu.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. To overcome these many impediments to gene replacement in Leishmania, we have developed a...

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Conditional knockout of Foxc2 gene in kidney: efficient generation of conditional alleles of single-exon gene by double-selection system

et al. 2015

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Foxc2 is a single-exon gene and a key regulator in development of multiple organs, including kidney. To avoid embryonic lethality of conventional Foxc2 knockout mice, we conditionally deleted Foxc2 in kidneys. Conditional targeting of a single-exon gene involves the large floxed gene segment spanning from promoter region to coding region to avoid functional disruption of the gene by the insertion of a loxP site. Therefore, in ES cell clones surviving a conventional single-selection, e.g., neomycin-resistant gene (neo) alone, homologous recombination between the long floxed segment and target genome results in a high incidence of having only one loxP site adjacent to the selection marker. To avoid this limitation, we employed a double-selection system. We generated a Foxc2 targeting construct in which a floxed segment contained 4.6 kb mouse genome and two different selection marker genes, zeocin-resistant gene and neo, that were placed adjacent to each loxP site. After double-selection by zeocin and neomycin, 72 surviving clones were screened that yielded three correctly targeted clones. After floxed Foxc2 mice were generated by tetraploid complementation, we removed the two selection marker genes by a simultaneous-single microinjection of expression vectors for Dre and Flp recombinases into in vitro-fertilized eggs. To delete Foxc2 in mouse kidneys, floxed Foxc2 mice were mated with Pax2-Cre mice. Newborn Pax2-Cre; Foxc2(loxP/loxP) mice showed kidney hypoplasia and glomerular cysts. These results indicate the feasibility of generating floxed Foxc2 mice by double-selection system and simultaneous removal of selection markers with a single microinjection.

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One-step generation of recombineering constructs by asymmetric-end ligation and negative selection

Cited by 6 publications

References 7 publications

Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression

Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression

A rapid, efficient and economical method for generating leishmanial gene targeting constructs

Conditional knockout of Foxc2 gene in kidney: efficient generation of conditional alleles of single-exon gene by double-selection system

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