Preparation and Testing of Synthetic mRNA for Microinjection

Moore, Wendy; Guille, Matt

doi:10.1385/1-59259-678-9:99

Cited by 4 publications

(3 citation statements)

References 21 publications

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“…Embryos were obtained as described by Smith and Slack ( 17 ) and staged according to Nieuwkoop and Faber ( 18 ). In vitro transcription and microinjection of mRNA was carried out as described previously ( 19 , 20 ). Injections of mRNA were carried out at the 1-cell stage with 4 nl of RNA solution (37 pg RNA total).…”

Section: Methodsmentioning

confidence: 99%

Identification of a structural and functional domain in xNAP1 involved in protein–protein interactions

Friedeberg¹,

Scarlett²,

McGeehan³

et al. 2006

Nucleic Acids Research

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xNAP1 (Xenopus nucleosome assembly protein) belongs to the family of nucleosome assembly proteins (NAPs) and shares 92% identity with human and mouse NAP1. NAPs have been reported to have a role in nucleosome assembly, cell cycle regulation, cell proliferation and transcriptional control, although the precise function of NAP1 is still not clear. Here we report the identification of a putative domain of xNAP1 by limited proteolysis. This domain has been mapped in the xNAP1 protein sequence to residues 38–282 and thus lacks the acidic sequences at the N- and C-termini. We have studied this domain and related fragments in vitro and by a functional assay involving over-expression of the protein in Xenopus laevis embryos. Analytical ultracentrifugation shows that removal of the acidic N- and C-terminal regions does not prevent the formation of larger multimers, which are predominantly hexadecamers. Injection of mRNA encoding the full-length xNAP1 or the putative domain and other related constructs into Xenopus embryos gave identical phenotypes. These results are discussed in relation to protein–protein interactions between NAP1 octamers and a possible ‘squelching’ mechanism.

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Section: Methodsmentioning

confidence: 99%

Identification of a structural and functional domain in xNAP1 involved in protein–protein interactions

Friedeberg¹,

Scarlett²,

McGeehan³

et al. 2006

Nucleic Acids Research

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“…To generate synthetic runx3 mRNA, the pCS2cmv‐ runx3 construct was digested with Not I and capped mRNA was synthesized in vitro by using the SP6 mMESSAGE mMACHINE Kit (Ambion) as described (Moore and Gullie, 1999). For rescue experiments, 10 pg of a synthetic mRNA was injected as described (Nasevicius and Ekker, 2000).…”

Section: Methodsmentioning

confidence: 99%

Runx3 is required for hematopoietic development in zebrafish

Kalev‐Zylinska

Horsfield

Flores

et al. 2003

Developmental Dynamics

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“…Embryos were obtained as described by Smith and Slack (1983) and staged according to Nieuwkoop and Faber (1967). RNA preparation and microinjection were carried out as described previously (Guille, 1999; Moore and Guille, 1999). Embryos at the two‐cell stage were injected with 4 nl of RNA solution, those at stage 6 were injected with 1 nl of RNase solution (50 ng of RNase) containing 10 mg/ml rhodamine dextran (+RNase) or 10 mg/ml rhodamine dextran alone (control).…”

Section: Methodsmentioning

confidence: 99%

RNA-dependent cytoplasmic anchoring of a transcription factor subunit duringXenopusdevelopment

et al. 2000

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contributed equally to this workThe CCAAT box transcription factor (CBTF) is a multimeric transcription factor that activates expression of the haematopoietic regulatory factor, GATA-2. The 122 kDa subunit of this complex, CBTF 122 , is cytoplasmic in fertilized Xenopus eggs and subsequently translocates to the nucleus prior to activation of zygotic GATA-2 transcription at gastrulation. Here we present data suggesting both a role for CBTF 122 prior to its nuclear translocation and the mechanism that retains it in the cytoplasm before the midblastula transition (MBT). CBTF 122 and its variant CBTF 98 are associated with translationally quiescent mRNP complexes. We show that CBTF 122 RNA binding activity is both necessary and suf®cient for its cytoplasmic retention during early development. The introduction of an additional nuclear localization signal to CBTF 122 is insuf®cient to overcome this retention, suggesting that RNA binding acts as a cytoplasmic anchor for CBTF 122 . Destruction of endogenous RNA by microinjection of RNase promotes premature nuclear translocation of CBTF 122 . Thus, the nuclear translocation of CBTF 122 at the MBT is likely to be coupled to the degradation of maternal mRNA that occurs at that stage.

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Preparation and Testing of Synthetic mRNA for Microinjection

Cited by 4 publications

References 21 publications

Identification of a structural and functional domain in xNAP1 involved in protein–protein interactions

Identification of a structural and functional domain in xNAP1 involved in protein–protein interactions

Runx3 is required for hematopoietic development in zebrafish

RNA-dependent cytoplasmic anchoring of a transcription factor subunit duringXenopusdevelopment

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