Simultaneous Gene Editing by Injection of mRNAs Encoding Transcription Activator-Like Effector Nucleases into Mouse Zygotes

Li, Chunliang; Qi, Rong; Singleterry, Rebecca; Hyle, Judith; Balch, Amanda; Li, Xiuling; Sublett, Jack; Berns, Hartmut; Valentine, Marcus B.; Valentine, Virginia; Sherr, Charles J.

doi:10.1128/mcb.00023-14

Cited by 26 publications

(18 citation statements)

References 47 publications

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“…The mosaicism was subsequently confirmed by sequencing of the Rosa26 locus in F1 generation mice (not shown). This indicates nuclease activity in later than the one-cell stage, as was recently described by Li and colleagues, who observed simultaneous gene editing in zygotes using TALENs [20]. This phenomenon could affect all nucleases Among Rosa26-TurboRFP founders, we identified 2 correctly targeted animals (R6 and R15) and one mouse with partial integration of the targeting vector (R8), which had lost a portion of the TurboRFP gene.…”

Section: Discussionsupporting

confidence: 74%

Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases

et al. 2014

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a b s t r a c tGene targeting in mice mainly employs homologous recombination (HR) in embryonic stem (ES) cells. Although it is a standard way for production of genetically modified mice, the procedure is laborious and time-consuming. This study describes targeting of the mouse Rosa26 locus by transcription activator-like effector nucleases (TALENs). We employed TALEN-assisted HR in zygotes to introduce constructs encoding TurboRFP and TagBFP fluorescent proteins into the first intron of the Rosa26 gene, and in this way generated two transgenic mice. We also demonstrated that these Rosa26-specific TALENs exhibit high targeting efficiency superior to that of zinc-finger nucleases (ZFNs) specific for the same targeting sequence. Moreover, we devised a reporter assay to assess TALENs activity and specificity to improve the quality of TALEN-assisted targeting.

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Section: Discussionsupporting

confidence: 74%

Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases

et al. 2014

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“…Continued breeding from these chimeric G1 H2A.B.3 --/x females with wt males (up to 4 generations), revealed that these additional mutations were lost after G2, but maintained original founder mutations (H2Afb3 X ∆+286 , gm14290 X ∆15 , H2Afb2 X ∆17 ). This can be explained by the fact that TALEN activity can be retained for several cell divisions following their injection into the 2--cell stage, thus producing a mosaic genotype, as previously observed 14,15 . Collectively, the G1 generation of the 9 founder Table 3).…”

Section: H2ab3 Genes (Supplementarymentioning

confidence: 68%

Gene editing of the multi-copy H2A.B gene family by a single pair of TALENS

Anuar

Field

Kurscheid

et al. 2017

Preprint

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In view of the controversy related to the generation of off-target mutations by gene editing approaches, we tested the specificity of TALENs by disrupting a multi-copy gene family using only one pair of TALENS. We show here that TALENS do display a high level of specificity by simultaneously knocking out the function of the three genes that encode for H2A.B.3. This represents the first described knockout of this histone variant.

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“…RNAs encoding forward and reverse TALEN nucleases directed to spacer sequences flanking the first Arf ATG codon were injected into C57BL/6J × FvN/N mouse zygotes and yielded mosaic pups in which a 25-bp deletion, including the 5′ ATG, generated a frame-shift mutation in which translation was initiated at M45 (19). A second strain with a single base-pair deletion immediately 3′ to the first ATG similarly initiated translation at M45 and was indistinguishable from the first.…”

Section: Generation Of Mouse Strains Expressing Either P19 Arf or P15mentioning

confidence: 99%

Small mitochondrial Arf (smArf) protein corrects p53-independent developmental defects of Arf tumor suppressor-deficient mice

Oosterwijk

Yang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The mouse p19 Arf (human p14 ARF ) tumor suppressor protein, encoded in part from an alternative reading frame of the Ink4a (Cdkn2a) gene, inhibits the Mdm2 E3 ubiquitin ligase to activate p53. Arf is not expressed in most normal tissues of young mice but is induced by high thresholds of aberrant hyperproliferative signals, thereby activating p53 in incipient tumor cells that have experienced oncogene activation. The single Arf mRNA encodes two distinct polypeptides, including full-length p19 Arf and N-terminally truncated and unstable p15 smArf ("small mitochondrial Arf") initiated from an internal in-frame AUG codon specifying methionine-45. Interactions of p19 Arf with Mdm2, or separately with nucleophosmin (NPM, B23) that localizes and stabilizes p19 Arf within the nucleolus, require p19 Arf N-terminal amino acids that are not present within p15 smArf . We have generated mice that produce either smARF alone or M45A-mutated (smArf-deficient) full-length p19 Arf proteins. BCR-ABL-expressing pro/pre-B cells producing smArf alone are as oncogenic as their Arf-null counterparts in generating acute lymphoblastic leukemia when infused into unconditioned syngeneic mice. In contrast, smArf-deficient cells from mice of the Arf M45A strain are as resistant as wild-type Arf +/+ cells to comparable oncogenic challenge and do not produce tumors. Apart from being prone to tumor development, Arf-null mice are blind, and their male germ cells exhibit defects in meiotic maturation and sperm production. Although Arf M45A mice manifest the latter defects, smArf alone remarkably rescues both of these p53-independent developmental phenotypes.Arf tumor suppressor | p53 | BCR-ABL acute lymphoblastic leukemia | spermatogenesis | hyaloid vasculature T hree exons (1α, 2, and 3) of the Cdkn2a (Ink4a) gene encode spliced mRNA segments that specify the Cdk4/6 inhibitor p16 Ink4a , whereas mRNA initiated from a distinct upstream 5′ exon (1β) opens an alternative reading in Ink4a exons 2 and 3 to yield p19 Arf (1), an inhibitor of the p53 ubiquitin ligase Mdm2 (2-5). As such, the unprecedented structure of the compact Ink4a-Arf locus, conserved in mammals, specifies two distinct tumor suppressors that interface with both the retinoblastoma protein (RB) and p53. The separate promoters upstream of exon-1α (encoding Ink4a) and exon-1β (encoding Arf) are induced by hyperproliferative signals generated by activated oncoproteins, triggering RB-and p53-dependent programs, respectively, that counter cellular self-renewal in response to oncogene stress (6, 7). Deletion or epigenetic silencing of the Ink4a-Arf locus coordinately diminishes the tumor suppressing effects of both RB and p53 and, as would be expected, inactivation of this gene cluster is one of the most frequently observed events in human cancer.The stability and nucleolar sequestration of p19 Arf depend on its interaction with nucleophosmin (NPM or B23), and Arf's ability to bind to either Mdm2 or NPM is determined by N-terminal amino acid residues encoded solely by exon-1β (8-11). Ind...

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Simultaneous Gene Editing by Injection of mRNAs Encoding Transcription Activator-Like Effector Nucleases into Mouse Zygotes

Cited by 26 publications

References 47 publications

Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases

Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases

Gene editing of the multi-copy H2A.B gene family by a single pair of TALENS

Small mitochondrial Arf (smArf) protein corrects p53-independent developmental defects of Arf tumor suppressor-deficient mice

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