Chapter 10 Manipulating Gene Expression with Replication--Competent Retroviruses

Morgan, Bruce; Fekete, Donna M.

doi:10.1016/s0091-679x(08)60629-9

Cited by 317 publications

(263 citation statements)

References 22 publications

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“…Epitope-tagged chimeric and wild-type α7 subunit cDNAs were first subcloned in frame into the adaptor plasmid Cla12 NCO 10,11 (provided by Dr. Donna Fekete) to add the 5' untranslated region of the src gene to the insert, which increases the levels of expressed protein obtained with RCASBP vectors, and to generate Cla1 ends 10 . Inserts were then cloned into the Cla1 site of RCASBP vector variants of the A-and Bsubgroup (provided by Dr. Donna Fekete) 9 , after the splice acceptor downstream from the constituitively active promoter in the viral long terminal repeat, and screened for proper orientation.…”

Section: Retroviral Vector-mediated Gene Transfer To Ciliary Ganglionmentioning

confidence: 99%

“…Inserts were then cloned into the Cla1 site of RCASBP vector variants of the A-and Bsubgroup (provided by Dr. Donna Fekete) 9 , after the splice acceptor downstream from the constituitively active promoter in the viral long terminal repeat, and screened for proper orientation. Viral stocks were prepared in chick embryo fibroblasts and concentrated to optimal titers for injecting, 5 x 10 7 to 10 9 , as described 10 .…”

Section: Retroviral Vector-mediated Gene Transfer To Ciliary Ganglionmentioning

confidence: 99%

“…To infect proliferating CG neuron precursors in the neural crest, RCASBP was injected into the mesencephalic region of the neural tube of St 9 White Leghorn chick embryos in ovo (Spafas), according to the protocol of Fekete 9,10 . Eggs were returned to the nonturning forced air-draft egg incubator at 37°C for 1-2 weeks to allow development to the stages of preganglionic synapse formation in the CG.…”

Section: Retroviral Vector-mediated Gene Transfer To Ciliary Ganglionmentioning

confidence: 99%

“…Chimeric subunits were generated by replacing the cytoplasmic loop of α7 with the loop of α3 or α5 to determine whether these added sequences could target extrasynaptic α7 to the synapse. The epitope-tagged chimeric subunits were overexpressed in CG neurons developing in vivo by using avian-specific replication-competent retroviral vectors (RCASBP) [9][10][11] . This retroviral vector stably integrates into the genome of dividing cells and leads to high levels of exogenous gene expression in the progeny throughout development.…”

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confidence: 99%

“…The myc-tagged chimeric subunits were overexpressed in developing CG neurons by using RCASBP vectors of the B-and A-envelope subgroups, RCASBP(B) either alone or together with RCASBP(A) to increase infection efficiency 9,10 . RCASBP containing the chimeric construct was injected into the mesencephalic region of the neural tube of stage (St) 9-10 chick embryos in ovo to optimally infect the dividing neural crest population that gives rise to CG neurons 14 .…”

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The long internal loop of the α3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo

et al. 1998

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ResultsUsing PCR, we constructed chimeric subunits in which the α7 cytoplasmic loop immediately after TM3 and before TM4 was replaced with the homologous region of the α3 or α5 sequence (Fig. 1a). The N-terminus up to TM2 regulates intersubunit assembly into receptor complexes, and α7 subunits do not coassemble with nAChR subunits, as demonstrated for endogenous subunits in CG neurons and for chimeric α7 subunits expressed in Xenopus laevis oocytes 4,5,12,13 . In particular, we have observed that coexpression of chimeric α7 subunits containing the α3 cytoplasmic loop together with wild-type α3 and β4 subunits in Xenopus oocytes results in the formation of two distinct receptor types that have the pharmacological properties of Bgt-nAChRs and nAChRs, respectively (data not shown). Thus, a difference in the distribution of chimeric α7 as compared to wild-type α7 on the infected CG neuron surface would result from the added α3 or α5 cytoplasmic loop. It cannot be explained by assembly with an endogenous nAChR subunit that can target to the synapse. Different types of neurotransmitter receptors coexist within single neurons and must be targeted to discrete synaptic regions for proper function. In chick ciliary ganglion neurons, nicotinic acetylcholine receptors (nAChRs) containing α3 and α5 subunits are concentrated in the postsynaptic membrane, whereas α-bungarotoxin receptors composed of α7 subunits are localized perisynaptically and excluded from the synapse. Using retroviral vector-mediated gene transfer in vivo, we show that the long cytoplasmic loop of α3 targets chimeric α7 subunits to the synapse and reduces endogenous nAChR surface levels, whereas the α5 loop does neither. These results show that a particular domain of one subunit targets specific receptor subtypes to the interneuronal synapse in vivo. Moreover, our findings suggest a difference in the mechanisms that govern assembly of interneuronal synapses as compared to the neuromuscular junction in vertebrates.1998 Nature America Inc.• http://neurosci.nature.com 1998 Nature America Inc.• http://neurosci.nature.com

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Section: Retroviral Vector-mediated Gene Transfer To Ciliary Ganglionmentioning

confidence: 99%

Section: Retroviral Vector-mediated Gene Transfer To Ciliary Ganglionmentioning

confidence: 99%

Section: Retroviral Vector-mediated Gene Transfer To Ciliary Ganglionmentioning

confidence: 99%

mentioning

confidence: 99%

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The long internal loop of the α3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo

et al. 1998

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High efficiency gene transfer into the embryonic chicken CNS using B-subgroup retroviruses

Homburger

Fekete

1996

Dev. Dyn.

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Successive formative stages of precartilaginous mesenchymal condensations in vitro: Modulation of cell adhesion by Wnt-7A and BMP-2

1999

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High-density chick limb bud cell culture is a useful model to study mesenchymal condensatifons and chondrogenesis. Most previous studies have focused on the effects of soluble reagents on terminal chondrogenic differentiation and have not defined the early cellular processes and signaling events. In this study, we defined five successive stages in the differentiation process: 1) dissociated cells, 2) small aggregates, 3) formation of cell clusters, 4) precartilaginous condensations, and 5) cartilage nodule. We used RCAS retrovirus-mediated Wnt-7a gene transduction to test the effect of Wnt-7a on the differentiation process. We found that Wnt-7a suppressed chondrogenic differentiation. Wnt-7a did not inhibit the initiation of condensation formation but blocked the progression of precartilaginous condensations to cartilage nodules. The Wnt-7a-transduced cultures showed characteristics of a less mature culture with persistent expression of NCAM, N-cadherin, wider distribution of integrin beta1 and fibronectin, and suppression of tenascin-C. BMP-2 is known to enhance chondrogenic differentiation in these cultures by promoting cell clusters to form continuous sheet-like precartilaginous condensations. However, cultures exposed to both BMP-2 and Wnt-7a showed inhibition of chondrogenic differentiation. Different signaling molecules such as Wnt-7a and BMP-2 may have antagonistic effects on cartilage differentiation and the gradient of the two molecules may be involved in defining the boundaries of the initial precartilaginous condensation. We propose that the shape of the precartilaginous condensations may be modulated by local concentrations of signaling molecules, such as Wnt-7a and BMP-2, which act to alter cell-substrate and cell-cell adhesions.

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Chapter 10 Manipulating Gene Expression with Replication--Competent Retroviruses

Cited by 317 publications

References 22 publications

The long internal loop of the α3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo

The long internal loop of the α3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo

High efficiency gene transfer into the embryonic chicken CNS using B-subgroup retroviruses

Successive formative stages of precartilaginous mesenchymal condensations in vitro: Modulation of cell adhesion by Wnt-7A and BMP-2

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