A rapid and non‐invasive selection of transgenic embryos before implantation using green fluorescent protein (GFP)

Ikawa, Masahito; Kominami, Katsuya; Yoshimura, Yayoi; Tanaka, Keiichi; Nishimune, Yoshitake; Okabe, Masaru

doi:10.1016/0014-5793(95)01162-8

Cited by 154 publications

(110 citation statements)

References 19 publications

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“…Clones, which proliferated in doxycycline-free medium but died in the presence of 1 g/ml doxycycline (Sigma-Aldrich) and 1 g/ml puromycin were selected as primary parental doxycycline-regulatory 3T3 cells. The open reading frame of enhanced GFP (EGFP) was amplified by polymerase chain reaction (PCR) by using LA-Taq polymerase (Takara Bio, Otsu, Japan) from pCX-GFP vector (Ikawa et al, 1995). Primers used were 5Ј-TGCCGACGCGTGCCACC ATGGTGAGCAAGG and 5Ј-ATAAGAATGCGGCCGCTGAGGAGTGAAT-TCTTACTT.…”

Section: Construction Of Inducible Gfp3t3 Fibroblast (Tre-gfp3t3) Cellsmentioning

confidence: 99%

Small Interfering RNA-mediated Silencing Induces Target-dependent Assembly of GW/P Bodies

Lian¹,

Fritzler

Katz³

et al. 2007

MBoC

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Gene silencing using small interfering RNA (siRNA) is a valuable laboratory tool and a promising approach to therapeutics for a variety of human diseases. Recently, RNA interference (RNAi) has been linked to cytoplasmic GW bodies (GWB). However, the correlation between RNAi and the formation of GWB, also known as mammalian processing bodies, remains unclear. In this report, we show that transfection of functional siRNA induced larger and greater numbers of GWB. This siRNA-induced increase of GWB depended on the endogenous expression of the target mRNA. Knockdown of GW182 or Ago2 demonstrated that the siRNA-induced increase of GWB required these two proteins and correlated with RNAi. Furthermore, knockdown of rck/p54 or LSm1 did not prevent the reassembly of GWB that were induced by and correlated with siRNA-mediated RNA silencing. We propose that RNAi is a key regulatory mechanism for the assembly of GWB, and in some cases, GWB may serve as markers for RNAi in mammalian cells. INTRODUCTIONGW bodies (GWB), also known as mammalian processing bodies (P bodies), are cytoplasmic foci that contain multiple decay factors and that are involved in the 5Ј33Ј mRNA degradation pathway. GWB are named from the marker protein GW182, which contains multiple glycine (G) and tryptophan (W) repeats and a classic RNA binding domain at the carboxyl terminus (Eystathioy et al., 2002a). The mRNA decay factors/complexes found in GWB include the deadenylase Ccr4, the decapping complex Dcp1a/1b/Dcp2, the LSm1-7 complex, Ge-1 (also known as Hedls), rck/p54, and exonuclease Xrn1 (Bashkirov et al., 1997;van Dijk et al., 2002;Ingelfinger et al., 2002;Lykke-Andersen, 2002;Eystathioy et al., 2003;Cougot et al., 2004;Andrei et al., 2005;Yu et al., 2005;Fenger-Gron et al., 2005). GWB are physically juxtaposed to and transiently interact with stress granules (SG). SG process cytoplasmic aggregates of stalled translational preinitiation complexes that accumulate during stress responses and that share certain components with GWB (Kedersha et al., 2005).In addition to mRNA decay, a crucial role of GWB and their components in RNA interference (RNAi) was recently uncovered (Anderson and Kedersha, 2006;Eulalio et al., 2007a;Jakymiw et al., 2007). RNAi is a posttranscriptional gene silencing mechanism that uses specific doublestranded RNA to silence genes in a sequence-specific manner Mello and Conte, 2004). In brief, the double-stranded RNA is processed by Dicer into small interfering RNA (siRNA) or microRNA (miRNA). The 21-to 26-nucleotide siRNA and miRNA are then incorporated in the effector complex, RNA-induced silencing complex (RISC), which either cleaves or inhibits translation of the target mRNA. In 2005, two key components of RISC, Argonaute2 (Ago2) and siRNA/miRNA, were found to be enriched in GWB (Sen and Blau, 2005;Pillai et al., 2005;Jakymiw et al., 2005;Liu et al., 2005b;Pauley et al., 2006). miRNA-targeted mRNA also localizes to GWB in a miRNAdependent manner (Liu et al., 2005b). These observations provide the first evidence that RNAi is ...

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Section: Construction Of Inducible Gfp3t3 Fibroblast (Tre-gfp3t3) Cellsmentioning

confidence: 99%

Small Interfering RNA-mediated Silencing Induces Target-dependent Assembly of GW/P Bodies

Lian¹,

Fritzler

Katz³

et al. 2007

MBoC

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“…Wall (1997) also points out that because plasmids may remain unincorporated into the genome and can segregate during cell division (producing mosaic embryos), that the ideal gfp reporter would somehow signal its incorporation into the genome. Of various strategies that have been used to identify transgenic/mosaic embryos prior to transfer to the recipients (Bowen et al, 1994;Ikawa et al, 1995;Chan et al, 1999) none preclude the identification of false positives due to the persistence of non-integrated microinjected DNA until the late stages of development. The loss of potential transgenic offspring may be due to the false negatives in transgenic mosaicism.…”

Section: The Incidence Of Mosaicism In Transgenic Embryosmentioning

confidence: 99%

“…At present, many authors have used the reporter gene technique in the preimplantation-stage screening protocol of many species of animals produced via pronuclear microinjection (Ikawa et al, 1995;Takada et al, 1997;Kato et al, 1999). This procedure allows the identification of presumptive transgenic embryos that can be transferred into surrogate recipients, thereby increasing the overall efficiency and reducing the cost of transgenic animal production (Keiser et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

The use of green ﬂuorescent protein (GFP) to select bovine embryos

Duszewska¹,

Козикова²,

Szydlik³

et al. 2003

J. Anim. Feed Sci.

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A major factor limiting the transgenesis in domestic animals is the inefficiency of maintaining large numbers of recipients carrying nontransgenic foetuses. The objectives of this study were: 1. to determine the influence of green fluorescent protein (GFP) construct injection on the development of bovine embryos, 2. to identify and select the GFP positive bovine embryos, and 3. to determine the rate of mosaicism in transgenic embryos. Cattle oocytes were matured and fertilised in vitro and zygotes were microinjected with pCX-EGFP construct consisting of CMV-IE enhancer, chicken β-actin promoter, cDNA of GFP (EGFP-732 bp) and rabbit β-globin polyadenylation sequences. Embryos from control (64) and microinjected (198) groups were cultured in vitro. After 168 h of culture, morula and blastocysts were observed in 39.06% of control and in 23.23% of injected group. We obtained three GFP positive embryos (1.51% of injected zygotes and 6.52% of morulae/blastocysts). One of them was 100, second 75 and third 25% GFP positive (66.7% of mosaicism). Use of gfp gene reporter to select bovine embryos is useful method to increase transgenic offspring, because GFP marker allows to choice only transgenic embryos and transfer them to recipients.

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“…GFP has been shown to be useful as a marker in transgenic mice given its lack of toxicity, high sensitivity, and reproducibility (31,32). We co-injected two types of DNA fragments, F 1 ␥ mutant minigene and GFP cDNA.…”

Section: Gene Expression Pattern Of Cag Promoter In Various Organsmentioning

confidence: 99%

Differential Regulation of Exonic Regulatory Elements for Muscle-specific Alternative Splicing during Myogenesis and Cardiogenesis

Ichida¹,

Hakamata²,

Hayakawa³

et al. 2000

Journal of Biological Chemistry

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Muscle-specific isoform of the mitochondrial ATP synthase ␥ subunit (F 1 ␥) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novo protein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem. 273, 8492-8501; Hayakawa, M., Endo, H., Hamamoto, T., and Kagawa, Y. (1998) Biochem. Biophys. Res. Commun. 251, 603-608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F 1 ␥ gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F 1 ␥ wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F 1 ␥ Pudel and F 1 ␥ Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum-or acid-stimulated splicing induction system in mouse myoblasts, Pudel mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F 1 ␥ Pu-del minigene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F 1 ␥ Pu-rich minigene mice revealed that the F 1 ␥ Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F 1 ␥ pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.Alternative pre-mRNA splicing is a fundamental process in eukaryotes that contributes to tissue-specific and developmentally regulated patterns of gene expression at the posttranscriptional level. Recently, both cis-acting elements and transacting factors have been reported to varying degrees (1-3). Significant progress has been made in identifying the alternative splicing mechanism involved in the Drosophila sex determination pathway (4 -7).Exonic and intronic cis-acting regulatory elements for RNA splicing have been reported in a number of mammalian genes located near weak 5Ј and 3Ј splice sites, and these elements appear to be involved in the control of stage-or tissue-specific splicing events (5, 8 -13). For example, a majority of exonic splicing enhancer (ESE) 1 elements have been reported to be abundant in purine nucleotides (14,15), ...

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A rapid and non‐invasive selection of transgenic embryos before implantation using green fluorescent protein (GFP)

Cited by 154 publications

References 19 publications

Small Interfering RNA-mediated Silencing Induces Target-dependent Assembly of GW/P Bodies

Small Interfering RNA-mediated Silencing Induces Target-dependent Assembly of GW/P Bodies

The use of green ﬂuorescent protein (GFP) to select bovine embryos

Differential Regulation of Exonic Regulatory Elements for Muscle-specific Alternative Splicing during Myogenesis and Cardiogenesis

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