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

Homburger, Sheila A.; Fekete, Donna M.

doi:10.1002/(sici)1097-0177(199605)206:1<112::aid-aja10>3.0.co;2-7

Cited by 37 publications

(17 citation statements)

References 24 publications

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“…APC::PSD93-dn cDNA [amino acids 2185-2232; corresponding to the APC C-terminus-end fragment that contains the consensus PDZ-binding motif (VTSV) at the 3' end] was similarly generated and HA-tagged. The cDNA constructs were subcloned separately into avian-specific retroviral vector RCASBP (B envelope subgroup type) (Homburger and Fekete, 1996). Viral stocks were prepared in DF1 chicken fibroblast cells (American Type Culture Collection, Manassas, VA).…”

Section: Retroviral Vector-mediated Gene Transfermentioning

confidence: 99%

Adenomatous polyposis coli plays a key role, in vivo, in coordinating assembly of the neuronal nicotinic postsynaptic complex

Rosenberg

Yang

Giovanni

et al. 2008

Molecular and Cellular Neuroscience

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The neuronal nicotinic synapse plays a central role in normal cognitive and autonomic function. Molecular mechanisms that direct the assembly of this synapse remain poorly defined, however. We show here that adenomatous polyposis coli (APC) organizes a multi-molecular complex that is essential for targeting α3*nAChRs to synapses. APC interaction with microtubule plus-end binding protein EB1 is required for α3*nAChR surface membrane insertion and stabilization. APC brings together EB1, the key cytoskeletal regulators macrophin and IQGAP1, and 14-3-3 adapter protein at nicotinic synapses. 14-3-3, in turn, links the α3-subunit to APC. This multi-molecular APC complex stabilizes the local microtubule and F-actin cytoskeleton and links postsynaptic components to the cytoskeleton-essential functions for controlling the molecular composition and stability of synapses. This work identifies macrophin, IQGAP1 and 14-3-3 as novel nicotinic synapse components and defines a new role for APC as an in vivo coordinator of nicotinic postsynaptic assembly in vertebrate neurons.

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Section: Retroviral Vector-mediated Gene Transfermentioning

confidence: 99%

Adenomatous polyposis coli plays a key role, in vivo, in coordinating assembly of the neuronal nicotinic postsynaptic complex

Rosenberg

Yang

Giovanni

et al. 2008

Molecular and Cellular Neuroscience

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“…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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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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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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Retrovirus Infection of Cells In Vitro and In Vivo

Cepko

Pear

1996

CP Molecular Biology

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There are many applications in which retrovirus vectors are used as transduction agents. In some cases, the vector carries a gene that one wishes to express in a target cell in order to study the function of that gene. In other cases, the virus is used to introduce a histochemical marker gene into cells in order to follow their fate. Retrovirus vectors can also be used in a variety of cells type to investigate regulatory sequences in which a reporter gene and regulatory sequences are carried by the vector and to immortalize or transform primary cells by transduction of oncogenes. For each application, the infection protocol may vary and must often be optimized. Guidelines for infection of cells in some typical in vivo and in vitro experiments are presented in this overview.

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

Cited by 37 publications

References 24 publications

Adenomatous polyposis coli plays a key role, in vivo, in coordinating assembly of the neuronal nicotinic postsynaptic complex

Adenomatous polyposis coli plays a key role, in vivo, in coordinating assembly of the neuronal nicotinic postsynaptic complex

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

Retrovirus Infection of Cells In Vitro and In Vivo

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