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A Phox2b BAC Transgenic Rat Line Useful for Understanding Respiratory Rhythm Generator Neural Circuitry.

Ikeda K, Takahashi M, Sato S, Igarashi H, Ishizuka T, Yawo H, Arata S, Southard-Smith EM, Kawakami K, Onimaru H - PLoS ONE (2015)

Bottom Line: Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG.In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies.Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; Division of Biology, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan.

ABSTRACT
The key role of the respiratory neural center is respiratory rhythm generation to maintain homeostasis through the control of arterial blood pCO2/pH and pO2 levels. The neuronal network responsible for respiratory rhythm generation in neonatal rat resides in the ventral side of the medulla and is composed of two groups; the parafacial respiratory group (pFRG) and the pre-Bötzinger complex group (preBötC). The pFRG partially overlaps in the retrotrapezoid nucleus (RTN), which was originally identified in adult cats and rats. Part of the pre-inspiratory (Pre-I) neurons in the RTN/pFRG serves as central chemoreceptor neurons and the CO2 sensitive Pre-I neurons express homeobox gene Phox2b. Phox2b encodes a transcription factor and is essential for the development of the sensory-motor visceral circuits. Mutations in human PHOX2B cause congenital hypoventilation syndrome, which is characterized by blunted ventilatory response to hypercapnia. Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG. In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies. Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.

No MeSH data available.


Related in: MedlinePlus

Schematic structure of the Phox2b-EYFP-2A-CreERT2 Rec BAC transgene.The exon-intron structure of the Phox2b gene is shown in the top section. The first exon which contains noncoding (white rectangle) and coding (black rectangle) regions, the second exon (black rectangle), and the third exon which contains coding (black rectangle) and noncoding (white rectangle) regions, are indicated as I, II, and III, respectively. The recombination strategy targeted the first exon (around the ATG site of Phox2b gene) inserting a cassette containing the enhanced yellow fluorescent protein (EYFP), 2A peptide coding sequence with fusion of Cre recombinase plus a modified tamoxifen-inducible estrogen receptor (CreERT2) sequence, and the SV40 polyadenylation sequence.
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pone.0132475.g002: Schematic structure of the Phox2b-EYFP-2A-CreERT2 Rec BAC transgene.The exon-intron structure of the Phox2b gene is shown in the top section. The first exon which contains noncoding (white rectangle) and coding (black rectangle) regions, the second exon (black rectangle), and the third exon which contains coding (black rectangle) and noncoding (white rectangle) regions, are indicated as I, II, and III, respectively. The recombination strategy targeted the first exon (around the ATG site of Phox2b gene) inserting a cassette containing the enhanced yellow fluorescent protein (EYFP), 2A peptide coding sequence with fusion of Cre recombinase plus a modified tamoxifen-inducible estrogen receptor (CreERT2) sequence, and the SV40 polyadenylation sequence.

Mentions: The Phox2b-EYFP-2A-CreERT2 Rec BAC construct was prepared by integrating coding sequences for a fluorescent protein and a CreERT2 tethered by 2A peptide into clone 95M11 derived from the CHORI RP24 C57BL/6J (B6) mouse genomic library (Fig 2). The backbone of RP24-95M11 is a BAC clone and contains the entire mouse Phox2b gene locus with no other neighboring genes (Fig 1). The RP24-95M11 BAC clone had been used previously to recapitulate the expression of the endogenous Phox2b gene in transgenic mice [53]. Because of the high homology between the mouse and rat Phox2b gene that extended into associated flanking regions, we expected that the RP24-95M11 BAC contained sufficient regulatory regions required to confer expression patterns of Phox2b gene in the rat during development and in adults. Similar findings were observed in two different lines that harbor tandemly-arrayed 30 copies of the clones.


A Phox2b BAC Transgenic Rat Line Useful for Understanding Respiratory Rhythm Generator Neural Circuitry.

Ikeda K, Takahashi M, Sato S, Igarashi H, Ishizuka T, Yawo H, Arata S, Southard-Smith EM, Kawakami K, Onimaru H - PLoS ONE (2015)

Schematic structure of the Phox2b-EYFP-2A-CreERT2 Rec BAC transgene.The exon-intron structure of the Phox2b gene is shown in the top section. The first exon which contains noncoding (white rectangle) and coding (black rectangle) regions, the second exon (black rectangle), and the third exon which contains coding (black rectangle) and noncoding (white rectangle) regions, are indicated as I, II, and III, respectively. The recombination strategy targeted the first exon (around the ATG site of Phox2b gene) inserting a cassette containing the enhanced yellow fluorescent protein (EYFP), 2A peptide coding sequence with fusion of Cre recombinase plus a modified tamoxifen-inducible estrogen receptor (CreERT2) sequence, and the SV40 polyadenylation sequence.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492506&req=5

pone.0132475.g002: Schematic structure of the Phox2b-EYFP-2A-CreERT2 Rec BAC transgene.The exon-intron structure of the Phox2b gene is shown in the top section. The first exon which contains noncoding (white rectangle) and coding (black rectangle) regions, the second exon (black rectangle), and the third exon which contains coding (black rectangle) and noncoding (white rectangle) regions, are indicated as I, II, and III, respectively. The recombination strategy targeted the first exon (around the ATG site of Phox2b gene) inserting a cassette containing the enhanced yellow fluorescent protein (EYFP), 2A peptide coding sequence with fusion of Cre recombinase plus a modified tamoxifen-inducible estrogen receptor (CreERT2) sequence, and the SV40 polyadenylation sequence.
Mentions: The Phox2b-EYFP-2A-CreERT2 Rec BAC construct was prepared by integrating coding sequences for a fluorescent protein and a CreERT2 tethered by 2A peptide into clone 95M11 derived from the CHORI RP24 C57BL/6J (B6) mouse genomic library (Fig 2). The backbone of RP24-95M11 is a BAC clone and contains the entire mouse Phox2b gene locus with no other neighboring genes (Fig 1). The RP24-95M11 BAC clone had been used previously to recapitulate the expression of the endogenous Phox2b gene in transgenic mice [53]. Because of the high homology between the mouse and rat Phox2b gene that extended into associated flanking regions, we expected that the RP24-95M11 BAC contained sufficient regulatory regions required to confer expression patterns of Phox2b gene in the rat during development and in adults. Similar findings were observed in two different lines that harbor tandemly-arrayed 30 copies of the clones.

Bottom Line: Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG.In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies.Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; Division of Biology, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan.

ABSTRACT
The key role of the respiratory neural center is respiratory rhythm generation to maintain homeostasis through the control of arterial blood pCO2/pH and pO2 levels. The neuronal network responsible for respiratory rhythm generation in neonatal rat resides in the ventral side of the medulla and is composed of two groups; the parafacial respiratory group (pFRG) and the pre-Bötzinger complex group (preBötC). The pFRG partially overlaps in the retrotrapezoid nucleus (RTN), which was originally identified in adult cats and rats. Part of the pre-inspiratory (Pre-I) neurons in the RTN/pFRG serves as central chemoreceptor neurons and the CO2 sensitive Pre-I neurons express homeobox gene Phox2b. Phox2b encodes a transcription factor and is essential for the development of the sensory-motor visceral circuits. Mutations in human PHOX2B cause congenital hypoventilation syndrome, which is characterized by blunted ventilatory response to hypercapnia. Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG. In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies. Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.

No MeSH data available.


Related in: MedlinePlus