CHT- knock-out mice have a loxP-flanked EGFP gene replacing part of the coding region, including the ATG initiation codon, of the Slc5a7 gene. These mice may be useful for studying cholinergic neurotransmission.
Randy D. Blakely, Vanderbilt University School of Medicine
Genetic Background | Generation |
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|
Allele Type | Gene Symbol | Gene Name |
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Targeted (Reporter, Null/Knockout) | Slc5a7 | solute carrier family 5 (choline transporter), member 7 |
CHT- knock-out mice have a loxP-flanked EGFP gene replacing part of the coding region, including the ATG initiation codon, of the solute carrier family 5 (choline transporter), member 7 (Slc5a7) gene. EGFP is detectable by immunostaining. SLC5A7 (CHT) is a Na(+)-dependent high-affinity transporter that mediates the uptake of choline for acetylcholine (ACh) synthesis in cholinergic neurons. ACh is a neurotransmitter released at the neuromuscular junction (NMJ), as well as at autonomic synapses, of the central and peripheral nervous system. ACh regulates a variety of autonomic, cognitive, and motor functions, and also plays a role in the development and maturation of both presynaptic and postsynaptic aspects of the NMJ. Deficits in ACh synthesis or response have been associated with myasthenic syndromes, while degeneration of the cholinergic neurons of the basal forebrain has been associated with the onset of Alzheimer’s Disease. Heterozygotes CHT+/- mice are viable and fertile, while homozygotes die within an hour after birth due to hypoxia caused by herniation of the diaphragm. Choline uptake and ACh synthesis are lost in the forebrain and hindbrain of CHT-/- mice. The NMJs of the mice exhibit developmental and neurotransmission defects.
Adult CHT+/- mice overcome reductions in CHT protein levels and sustain choline uptake at wild-type levels through posttranslational mechanisms. These heterozygous mice display impaired physical performance with a slower rate of recovery compared to wildtype mice. They also have reduced brain ACh levels, elevated choline levels, and brain region-specific decreased expression of muscarinic acetylcholine receptors. They exhibit age-dependent ventricular dysfunction, as well as tachycardia and hypertension at rest.
A targeting vector was designed by Dr. Randy Blakely to replace part of the coding region, including the ATG in exon two and extending into intron 4, of the solute carrier family 5 (choline transporter), member 7 (Slc5a7) gene with a loxP-flanked enhanced green fluorescent protein (EGFP) gene and a frt-flanked neomycin resistance (neo) cassette. The construct was electroporated into 129S6/SvEvTac-derived TL1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts and the resulting chimeric males were bred to C57BL/6 females. Offspring were bred to Flp recombinase expressing mice on a C57BL/6 background to remove the neo cassette, and progeny were crossed to remove the Flp-expressing transgene. CHT- mice were backcrossed to C57BL/6NHsd mice for 12 generations and then were bred to C57BL/6J for at least 6 generations. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
Expressed Gene | GFP, Green Fluorescent Protein, |
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Site of Expression | Cholinergic neurons. |
Allele Name | targeted mutation 1.1, Randy D Blakely |
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Allele Type | Targeted (Reporter, Null/Knockout) |
Allele Synonym(s) | |
Gene Symbol and Name | Slc5a7, solute carrier family 5 (choline transporter), member 7 |
Gene Synonym(s) | |
Expressed Gene | GFP, Green Fluorescent Protein, |
Site of Expression | Cholinergic neurons. |
Strain of Origin | 129S6/SvEvTac |
Chromosome | 17 |
Molecular Note | The targeting vector was designed to replace part of the coding region, including the ATG in exon 2 and extending into intron 4, of the gene with a loxP-flanked enhanced green fluorescent protein (EGFP) gene and an FRT-flanked neomycin resistance (neo) cassette. Flp-mediated recombination removed the FRT-flanked neo cassette. |
When maintaining a live colony, heterozygous mice may be bred to wildtype mice from the colony, or to C57BL/6J inbred mice (Stock No. 000664). Homozygous mice do not survive past the first hour after birth due to hypoxia caused by herniation of the diaphragm.
When using the CHT- mouse strain in a publication, please cite the originating article(s) and include JAX stock #028692 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
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Herterozygous or wildtype for Slc5a7<tm1.1Rbl> |
Frozen Mouse Embryo | B6.129S6(Cg)-Slc5a7<tm1.1Rbl>/J | $2595.00 |
Frozen Mouse Embryo | B6.129S6(Cg)-Slc5a7<tm1.1Rbl>/J | $2595.00 |
Frozen Mouse Embryo | B6.129S6(Cg)-Slc5a7<tm1.1Rbl>/J | $3373.50 |
Frozen Mouse Embryo | B6.129S6(Cg)-Slc5a7<tm1.1Rbl>/J | $3373.50 |
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