A single point mutation activates the Moloney murine leukemia virus long terminal repeat in embryonal stem cells

Grez, Manuel; Zörnig, Martin; Nowock, Joachim; Ziegler, Marion

doi:10.1128/jvi.65.9.4691-4698.1991

Cited by 57 publications

(22 citation statements)

References 36 publications

(50 reference statements)

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“…There have been numerous investigations of the identity and effects of mutations that expand host range (Grez et al 1991;Subbarao et al 1993;Morris et al 1994;Shioda et al 1994;Kozak and Chakraborti 1996;Llamas-Saiz et al 1996;Doi et al 1997;Jonah et al 2003;Duffy et al 2006). However, several characteristics of these investigations limit their ability to predict equilibrium frequencies of potential colonists in natural populations.…”

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confidence: 99%

High Frequency of Mutations That Expand the Host Range of an RNA Virus

2007

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The ability of a virus population to colonize a novel host is predicted to depend on the equilibrium frequency of potential colonists (i.e., genotypes capable of infecting the novel host) in the source population. In this study, we investigated the determinants of the equilibrium frequency of potential colonists in the RNA bacteriophage f6. We isolated 40 spontaneous mutants capable of infecting a novel Pseudomonas syringae host and sequenced their host attachment genes to identify the responsible mutations. We observed 16 different mutations in the host attachment gene and used a new statistical approach to estimate that 39 additional mutations were missed by our screen. Phenotypic and fitness assays confirmed that the proximate mechanism underlying host range expansion was an increase in the ability to attach to the novel host and that acquisition of this ability most often imposed a cost for growth rate on two standard hosts. Considered in a population genetic framework, our data suggest that host range mutations should exist in phage populations at an equilibrium frequency (3 3 10 À4) that exceeds the phage mutation rate by more than two orders of magnitude. Thus, colonization of novel hosts is unlikely to be limited by an inability to produce appropriate mutations.

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confidence: 99%

High Frequency of Mutations That Expand the Host Range of an RNA Virus

2007

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“…The inactivity of the enhancer is mediated by its interaction with negatively acting cellular factors (1,16,28,41,42). A variant LTR was isolated from the myeloproliferative sarcoma virus (MPSV) (8) and shown to express more strongly than the MoMuLV LTR in EC cells (17,19,23,42) and in hematopoietic cells (3,29,36). The fundamental difference between the MoMuLV and the MPSV enhancer repeats is the presence in MPSV of a consensus site for binding the transcription factor Sp1 (31).…”

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confidence: 99%

“…An endogenous murine retrovirus, dl587rev, was isolated from murine genomic sequences and found to contain a novel PBS sequence which includes adenine at position ϩ160 (10). Inclusion of the dl587rev PBS in retroviruses allowed increased expression in EC cells compared with the wild-type MoMuLV PBS (1,17).…”

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confidence: 99%

“…However, there is evidence that the MoMuLV enhancer and PBS may have negative effects even on heterologous promoters placed internally (2,16). Another approach involves incorporation of various sequence elements which are active in ES cells, to replace the MoMuLV sequences, such as the enhancer from the MPSV variant (19) or from a mutant polyomavirus (23), or the PBS from dl587rev (17,42). These attempts had limited success, perhaps because of the interaction of multiple inhibitory elements in or near the LTR.…”

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confidence: 99%

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Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells

Challita

Skelton

El-Khoueiry

et al. 1995

J Virol

201

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Infection by murine retroviruses in embryonic carcinoma (EC) and embryonic stem cells is highly restricted. The transcriptional unit of the Moloney murine leukemic virus (MoMuLV) long terminal repeat (LTR) is inactive in EC and embryonic stem cells in association with increased proviral methylation. In this study, expression in F9 EC cells was achieved from novel retroviral vectors containing three modifications in the MoMuLV-based retroviral vector: presence of the myeloproliferative sarcoma virus LTR, substitution of the primer binding site, and either deletion of a negative control region at the 5 end of the LTR or insertion of a demethylating sequence. We conclude that inhibition of expression from the MoMuLV LTR in EC cells is mediated through the additive effects of multiple cis-acting elements affecting the state of methylation of the provirus.), and the G1Na plasmid was provided by P. Tolstechev (Genetic Therapy, Inc., Gaithersburg, Md.). The LN vector was constructed and packaged in the laboratory of A. Dusty Miller (Fred Hutchinson Cancer Center, Seattle, Wash.).The MPSV LTR was used to replace the 3Ј MoMuLV LTR of G1Na to make MPneo. The NCR was removed from the MPSV LTR as an NheI (at nucleotide 33 in the LTR)-to-Sau3a (at nucleotide 97 in the LTR) fragment. The cut ends of the LTR were ligated together after fill-in by Klenow DNA polymerase to make the MPncr 3Ј LTR; this was then used to replace the 3Ј LTR of G1Na, yielding MPncrneo. The Thy-1 fragment in the plasmid Bluescript was opened at the SmaI site immediately 3Ј of the insert, and a synthetic oligonucleotide encoding an XbaI site (New England Biolabs, Beverly, Mass.) was ligated in place. The Thy-1 piece was isolated as an XbaI-XbaI fragment and cloned into the NheI site of the MPSV LTR in Bluescript. The Thy-1-substituted MPSV LTR was inserted in place of the 3Ј LTR in G1Na to make MPthyneo.The 5Ј LTR and psi region from plasmid LN was subcloned as an EcoRI-EcoRI fragment. The KpnI-SpeI fragment encompassing the PBS was removed and replaced by the KpnI-SpeI fragment from dl587rev. The 5Ј LTR/leader region fragment containing the dl587rev PBS was then returned to the MPneo, MPthyneo, and MPncrneo plasmids to produce MPdlneo, MPthydlneo, and MPncrdlneo.To make LNncrneo, the NCR (NheI at position 33 to Sau3a at position 97; Fig.

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“…Secondly, a negatively acting cis ‐element, referred to as the negative control region, which binds the cellular factor YY1 that contributes to transcriptional repression [28,29], was deleted. Thirdly, by replacing the primer binding site (PBS) with the PBS from an endogenous murine retrovirus, dl587rev, another inhibitory element that binds a cellular transcription inhibitor, was eliminated [30].…”

Section: In Vitro Human Fviii Expression In Human Bm Mesenchymal Cellmentioning

confidence: 99%

Bone marrow mesenchymal cells for haemophilia A gene therapy using retroviral vectors with modified long‐terminal repeats

et al. 2003

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Bone marrow (BM) cells are attractive target cells for ex vivo gene therapy of genetic diseases, including haemophilia A. However, BM-derived haematopoietic stem/progenitor cells (HSCs) transduced with factor VIII (FVIII) retroviral vectors, failed to express FVIII in vivo. To overcome the limitations of HSCs for haemophilia gene therapy, BM-derived mesenchymal cells were explored as alternative target cells. The BM mesenchymal cell population contains self-renewing mesenchymal stem/progenitor cells that give rise to different mesenchymal lineages and have been used safely in phase I gene-marking trials. Human BM mesenchymal cells were transduced in vitro with an improved retroviral vector encoding a human B-domain deleted FVIII (hFVIIIdeltaB) cDNA (MND-MFG-hFVIIIdeltaB). This vector contains multiple modifications in the cis-acting elements within the MoMLV long-terminal repeats (LTR) that prevent the binding of repressive transcription factors. These modifications were previously shown to increase and prolong gene expression in embryonic stem (ES) cells and HSCs. Transduction of BM mesenchymal cells with the MND-MFG-hFVIIIdeltaB retroviral vector resulted in high levels of functional human FVIII in vitro, ranging between 300 +/- 50 SD and 700 +/- 100 SD mU per 106 cells per 24 h. Following xenografting of the transduced human BM cells into immunodeficient NOD-SCID mice, therapeutic hFVIII levels of 12 +/- 10 ng mL-1 were detected in the plasma. Polymerase chain reaction analysis demonstrated long-term engraftment (>3 months) of the human BM mesenchymal cells. The long-term persistence of BM mesenchymal cells in the absence of myelo-ablative conditioning and the therapeutic FVIII levels in vivo underscore the potential usefulness of BM-derived mesenchymal cells for haemophilia gene therapy, as opposed to BM-derived HSCs. Despite the modifications of the MoMLV LTR, FVIII expression declined, which coincided with a decrease in FVIII mRNA transcription levels, indicating that the salutary effect of the LTR modification on transgene expression is not universally applicable to all cell types.

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A single point mutation activates the Moloney murine leukemia virus long terminal repeat in embryonal stem cells

Cited by 57 publications

References 36 publications

High Frequency of Mutations That Expand the Host Range of an RNA Virus

High Frequency of Mutations That Expand the Host Range of an RNA Virus

Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells

Bone marrow mesenchymal cells for haemophilia A gene therapy using retroviral vectors with modified long‐terminal repeats

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