[51] Gene targeting in embryonic stem cells

Ramírez‐Solís, Ramiro; Davis, A C; Bradley, Allan

doi:10.1016/0076-6879(93)25054-6

Cited by 288 publications

(182 citation statements)

References 35 publications

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“…The targeting vector was linearized for transfection by using a NheI digest. AB2.2 (129S7/SvEvBrd-Hprtb-m2 ) ES cells were propagated, transfected by electroporation, and selected with G418 by using standard protocols (29). Homologous recombination was confirmed by using Southern blotting of EcoRI digested with 5Ј and 3Ј external probes amplified from the genomic PAC clone.…”

Section: Methodsmentioning

confidence: 99%

Prokineticin receptor 2 (Prokr2) is essential for the regulation of circadian behavior by the suprachiasmatic nuclei

Prosser

Bradley

Chesham

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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136

138

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The suprachiasmatic nucleus (SCN), the brain's principal circadian pacemaker, coordinates adaptive daily cycles of behavior and physiology, including the rhythm of sleep and wakefulness. The cellular mechanism sustaining SCN circadian timing is well characterized, but the neurochemical pathways by which SCN neurons coordinate circadian behaviors remain unknown. SCN transplant studies suggest a role for (unidentified) secreted factors, and one potential candidate is the SCN neuropeptide prokineticin 2 (Prok2). Prok2 and its cognate prokineticin receptor 2 (Prokr2/Gpcr73l1) are widely expressed in both the SCN and its neural targets, and Prok2 is light-regulated. Hence, they may contribute to cellular timing within the SCN, entrainment of the clock, and/or they may mediate circadian output. We show that a targeted null mutation of Prokr2 disrupts circadian coordination of the activity cycle and thermoregulation. Specifically, mice lacking Prokr2 lost precision in timing the onset of nocturnal locomotor activity; and under both a light/dark cycle and continuous darkness, there was a pronounced temporal redistribution of activity away from early to late circadian night. Moreover, the coherence of circadian behavior was significantly reduced, and nocturnal body temperature was depressed. Entrainment by light is not, however, dependent on Prokr2, and bioluminescence real-time imaging of organotypical SCN slices showed that the mutant SCN is fully competent as a circadian oscillator. We conclude that Prokr2 is not necessary for SCN cellular timekeeping or entrainment, but it is an essential link for coordination of circadian behavior and physiology by the SCN, especially in defining the onset and maintenance of circadian night.T he suprachiasmatic nucleus (SCN) of the hypothalamus constitutes an autonomous circadian pacemaker necessary for the maintenance of daily rhythms, including the cycle of sleep and wakefulness (1). The molecular basis of the cellular oscillator of the SCN is broadly characterized, but how the SCN signals circadian time to other sites in the brain and thence to peripheral tissues is unclear (2). Neuroanatomical projections from the SCN run predominantly to local hypothalamic and thalamic structures (3), including the dorsomedial nucleus (DMN), a principal site for the regulation of sleep and wakefulness and circadian arousal by behavioral cues (4, 5). How such ''hard-wired'' connections influence SCN targets remains open to question. Although electrical signaling by means of synaptic contacts is likely important (6), encapsulated SCN grafts unable to form synaptic contacts with host tissues are nevertheless able to establish circadian activity/rest cycles in previously arrhythmic, SCN-lesioned recipients (7). This finding indicates that paracrine factors from the SCN mediate circadian coordination within the brain. A small number of candidate factors have been explored, including TGF-␣ (8) and cardiotrophin-like cytokine (9). These factors are expressed by the SCN on a circadian basis, and local ...

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Section: Methodsmentioning

confidence: 99%

Prokineticin receptor 2 (Prokr2) is essential for the regulation of circadian behavior by the suprachiasmatic nuclei

Prosser

Bradley

Chesham

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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136

138

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“…The D79N mouse line was created using a hit and run gene targeting strategy (18), as described (8). Male chimeras were mated with C57BL͞6 mice to generate heterozygous mice for intercrosses.…”

Section: Methodsmentioning

confidence: 99%

Substitution of a mutant α _2a -adrenergic receptor via “hit and run” gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo

Lakhlani

MacMillan

Guo

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

315

234

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Norepinephrine contributes to antinociceptive, sedative, and sympatholytic responses in vivo, and ␣ 2 adrenergic receptor (␣ 2 AR) agonists are used clinically to mimic these effects. Lack of subtype-specific agonists has prevented elucidation of the role that each ␣ 2 AR subtype (␣ 2A , ␣ 2B , and ␣ 2C ) plays in these central effects. ␣ 2 -adrenergic receptors (␣ 2 ARs) present in the central nervous system (CNS) respond to norepinephrine (NE) and epinephrine and mediate sympatholytic, sedative-hypnotic, analgesic, anesthetic-sparing, hypotensive, and anxiolytic responses (1). Many of these responses are therapeutically useful and are exploited clinically, for example, during anesthesia and to attenuate the symptoms of opioid withdrawal (2). Three ␣ 2 AR subtypes have been revealed by pharmacological (␣ 2A AR, ␣ 2B AR, and ␣ 2C AR) and molecular cloning (␣ 2a AR, ␣ 2b AR, and ␣ 2c AR) strategies (3), and all couple, via pertussis toxin-sensitive G i ͞G o proteins, to attenuation of adenylyl cyclase, suppression of voltage-gated Ca 2ϩ channels, and activation of inwardly rectifying K ϩ channels (4).Multiple experimental limitations have precluded clarifying the involvement of each ␣ 2 AR subtype in catecholaminemediated physiological responses in the CNS. Subtype-specific ␣ 2 AR agonists and antagonists are not available (5); even when subtype selectivity has been noted in vitro, varying and unknown in vivo bioavailability precludes confident correlation of the administered dose with the amount of drug at the receptor site. Previous studies to explore ␣ 2 AR involvement in various responses have used prazosin to block catecholamine responses mediated by ␣ 1 adrenergic receptors (␣ 1 AR); however, it is now known that the ␣ 2B AR and ␣ 2C AR subtypes also are blocked by prazosin (5), thus confounding the interpretations of these earlier studies. In addition, because ␣ 1 AR can functionally antagonize ␣ 2 AR-mediated responses in some settings, ␣ 2 AR responses in the presence of prazosin (added to block ␣ 1 AR, ␣ 2B AR, and ␣ 2C AR) may reflect the disturbance of the balance between the functionally antagonistic ␣ 2 AR and ␣ 1 AR systems rather than provide insights concerning the role of the ␣ 2A AR subtype. Consequently, we manipulated the mouse genome to provide definitive evidence regarding the role of the ␣ 2A AR subtype in CNS responses.We used the ''hit and run'' targeting variant of homologous recombination (6, 7) to substitute a subtle mutation of the ␣ 2a AR, D79N into the mouse genome as a tool to explore the role of the ␣ 2a AR in vivo (8). The aspartate residue at position 79 (D79) is highly conserved in a topologically identical position in the second transmembrane span in a large subset of G protein-coupled receptors (9). Mutation of this residue has been shown to eliminate allosteric regulation of receptor binding by monovalent cations (10-13) and to perturb receptor-G protein-effector coupling (14-17) in heterologous expression systems. Thus, the animals expressing the D79N ␣ 2a AR provi...

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“…The gene targeting construct was constructed by subcloning a 1.1-kb XmnI-ScaI genomic fragment into the blunted XhoI site of pPNT and a 4.8-kb EcoRI fragment into the EcoRI site. The construct was linearized with NotI and electroporated into R1 embryonic stem (ES) cells by using protocols described by Ramirez-Solis et al (27). Two correctly targeted clones were isolated from 300 colonies screened.…”

Section: Methodsmentioning

confidence: 99%

A Targeted Mutation at the T-Cell Receptor α/δ Locus Impairs T-Cell Development and Reveals the Presence of the Nearby Antiapoptosis Gene Dad1

Hong

Cado

Mitchell

et al. 1997

Molecular and Cellular Biology

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Locus control regions are cis gene regulatory elements comprised of DNase I-hypersensitive sites. These regions usually do not stimulate transcription outside of a chromosomal context, and therefore their ability to regulate the expression of genes is thought to occur through the modification of chromatin accessibility. A locus control region is located downstream of the T-cell receptor (TCR) ␣/␦ locus on mouse chromosome 14. This locus control region is known to drive T-cell-specific TCR␣ transcription in transgenic mice. In this report, we describe a targeted deletion of this locus control region and show that this mutation acts at a critical checkpoint in ␣␤ T-cell development, between the TCR-intermediate and TCR-high stages. Our analysis further reveals that the antiapoptosis gene Dad1 is at the 3 end of the TCR ␣/␦ locus and that Dad1 is required for embryogenesis. We show that mouse Dad1 has a broader expression pattern than the TCR genes, in terms of both tissue and temporal specificity. Finally, we report that the chromatin between TCR␣ and Dad1 is DNase I hypersensitive in a variety of cell types, thus correlating with Dad1 expression and raising the possibility that Dad1 regulatory sequences reside in this region.

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[51] Gene targeting in embryonic stem cells

Cited by 288 publications

References 35 publications

Prokineticin receptor 2 (Prokr2) is essential for the regulation of circadian behavior by the suprachiasmatic nuclei

Prokineticin receptor 2 (Prokr2) is essential for the regulation of circadian behavior by the suprachiasmatic nuclei

Substitution of a mutant α _2a -adrenergic receptor via “hit and run” gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo

A Targeted Mutation at the T-Cell Receptor α/δ Locus Impairs T-Cell Development and Reveals the Presence of the Nearby Antiapoptosis Gene Dad1

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[51] Gene targeting in embryonic stem cells

Cited by 288 publications

References 35 publications

Prokineticin receptor 2 (Prokr2) is essential for the regulation of circadian behavior by the suprachiasmatic nuclei

Prokineticin receptor 2 (Prokr2) is essential for the regulation of circadian behavior by the suprachiasmatic nuclei

Substitution of a mutant α 2a -adrenergic receptor via “hit and run” gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo

A Targeted Mutation at the T-Cell Receptor α/δ Locus Impairs T-Cell Development and Reveals the Presence of the Nearby Antiapoptosis Gene Dad1

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Substitution of a mutant α _2a -adrenergic receptor via “hit and run” gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo