Strain Detail Sheet

Strain Name:
B6J.Cg-Chrna4tm4Lst Tg(Chrna6*)4Lst/Mmmh
Stock Number:
068051-MU
Citation ID:
RRID:MMRRC_068051-MU
Other Names:
CHRNA6GFP x CHRNA4MCHERRY

Strain Information

Gene Details

Chrna4tm4Lst
Name: cholinergic receptor, nicotinic, alpha polypeptide 4; targeted mutation 4, Henry Lester
Synonyms: alpha4-mCherry
Type: Allele
Species: Mus musculus (mouse)
Chromosome: 2
Alteration at locus: Knock-In
Chrna4
Name: cholinergic receptor, nicotinic, alpha polypeptide 4
Synonyms: alpha4 nAChR, a4 nicotinic receptor, Acra4, Acra-4, alpha4-nAChR
Type: Gene
Species: Mouse
Chromosome: 2
Alteration at locus: Knock-In
NCBI: 11438
HGNC: HGNC:1958
Homologene: 592
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Additional Resources
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mCherry
Name: red fluorescent fluorophore, mCherry
Type: Gene
Species: Discosoma sp. (mushroom coral)
Chromosome:
Alteration at locus: Knock-In
Tg(Chrna6*)4Lst
Name: transgene insertion 4, Henry A. Lester
Type: Allele
Species: Mus musculus (mouse)
Chromosome: unk
Alteration at locus: Knock-In
EGFP
Name: enhanced green fluorescent protein
Type: Gene
Species: Aequorea victoria (jellyfish)
Chromosome:
Alteration at locus: Knock-In
Genetic Alterations
Knock-in for one gene (CHRNA4-MCHERRY); BAC-transgenic for second gene (CHRNA6-GFP)
ES Cell Line
  • Chrna4tm4Lst allele: CJ7 derived from 129S1/Sv-Oca2+ Tyr+ Kitl+
  • Tg(Chrna6*)4Rmdr allele: N/A

Strain Description

Phenotype
Nicotinic Receptor subunits are tagged to fluorescent proteins. Alpha6 fused to GFP (under a bacterial artificial chromosome) and alpha4 fused to mCherry (traditional knock-in).
MeSH Terms
  • Animals
  • Behavior, Animal/drug effects
  • Conditioning, Psychological/drug effects
  • Dopaminergic Neurons/drug effects
  • Dopaminergic Neurons/metabolism
  • Electronic Nicotine Delivery Systems
  • Farnesol/pharmacology
  • Female
  • Flavoring Agents/pharmacology
  • GABAergic Neurons/drug effects
  • GABAergic Neurons/metabolism
  • Locomotion
  • Male
  • Mesencephalon/cytology
  • Mesencephalon/drug effects
  • Mesencephalon/metabolism
  • Mice
  • Microscopy, Confocal
  • Nicotine/pharmacology
  • Nicotinic Agonists/pharmacology
  • Patch-Clamp Techniques
  • Receptors, Nicotinic/metabolism
  • Reward
  • Sex Factors
  • Up-Regulation
  • Vaping/metabolism
  • Vaping/psychology
  • Ventral Tegmental Area/cytology
  • Ventral Tegmental Area/drug effects
  • Ventral Tegmental Area/metabolism
  • Action Potentials/drug effects
  • Action Potentials/physiology
  • Antipruritics/chemistry
  • Antipruritics/pharmacology
  • Cells, Cultured
  • Dopaminergic Neurons/physiology
  • Menthol/chemistry
  • Menthol/pharmacology
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Models, Molecular
  • Protein Binding/drug effects
  • Receptors, Nicotinic/genetics
  • Time Factors
  • Tyrosine 3-Monooxygenase/genetics
  • Tyrosine 3-Monooxygenase/metabolism
  • Up-Regulation/drug effects
  • Xenopus laevis
Strain Development
Construction of BAC Transgenic α6-GFP Mice (detailed fully in PMID: 22836257)
Injection-grade α6-GFP BAC DNA was prepared via double CsCl branding (Lofstrand labs). BAC DNA was injected into the pronucleus of fertilized B6D2F1 ova and implanted into pseudopregnant Swiss-Webster surrogates. Several transgenic founders (identified via tail biopsy) were crossed to C57BL/6J to obtain germline transmission and to establish a colony. These were continually backcrossed to C57BL/6J. For the founder lines, the α6-GFP copy number was calculated using real-time PCR. Backcrossed to C57BL/6J every time a new mating pair was established (and deemed B6J-congenic at the time of the allele combination).

Construction of α4-mCherry knock-in mice
The α4-mCherry knock-in mouse was constructed in a manner similar to the previously described α4-YFP knock-in mouse (PMID:17670967). Briefly, a α4-mCherry knock-in construct was linearized and electroporated into CJ7 mouse embryonic stem (ES) cells. Recombinant clones were selected with G418 and screened for the presence of the neo cassette and mCherry insert by a combination of PCR and automated sequence analysis. To confirm the correct position of the homologous recombination of the targeting construct within the genome and the insertion of mCherry and neo cassette, ES cells were screened by PCR and sequencing. Karyotype analysis was used to assess chromosome numbers. Positive clones with the highest percentage of the correct number of chromosomes were selected for a subsequent round of electroporation. Clones were injected into C57BL/6 blastocysts to create chimeras. Chimeras were mated with wild-type animals and germline transmission was established. Founder mice were mated and colonies with α4-mCherry homozygous mice were established. For timed mating of α4-mCherry homozygous mice, genotypes were assessed by PCR analysis of tail DNA. Backcrossed to C57BL/6J every 10th generation (and deemed congenic at the time of the allele combination).

Construction of α6-GFPα4-mCherry mice
To produce α4-mCherryα6-GFP mice, α4-mCherry knock-in mice, maintained at Caltech (PMID: 26961950 and 26740650), were first crossed to α6-GFP BAC transgenic mice (PMID: 22836257 and 24378908) to produce α4-mCherry+/-α6-GFP mice (heterozygous for α4-mCherry, transgenic for α6-GFP). α4-mCherry+/-α6-GFP mice were then crossed to α4-mCherry+/+ mice to produce α4-mCherry+/+α6-GFP mice. Mice of this genotype were continually backcrossed to α4-mCherry+/+ mice. The presence of α4-mCherry and α6-GFP was independently confirmed for each mouse using PCR. Maintenance via intra-strain mating.
Suggested Control Mice
Wild-type or C57BL/6J

MMRRC Genetic QC

MMRRC Genetic QC Summary
The MMRRC Centers have developed a genetic QC pipeline using MiniMUGA array genotyping to provide additional information on strain backgrounds for MMRRC congenic and inbred strains. For more information on when data may be available, or to request genotyping for a strain of interest, please contact mmrrc@missouri.edu. Older strains may not have this information.

Research Applications

  • Neurobiology

Strain Origin

Donor
Brandon Henderson, Ph.D., Joan C Edwards School of Medicine at Marshall University.
Primary Reference

Akers AT, Cooper SY, Baumgard ZJ, Casinelli GP, Avelar AJ, Henderson BJ. Upregulation of nAChRs and Changes in Excitability on VTA Dopamine and GABA Neurons Correlates to Changes in Nicotine-Reward-Related Behavior. eNeuro. 2020 Oct 15;7(5):ENEURO.0189-20.2020. doi: 10.1523/ENEURO.0189-20.2020. (Medline PMID: 32988984)

Cooper SY, Akers AT, Henderson BJ. Green Apple e-Cigarette Flavorant Farnesene Triggers Reward-Related Behavior by Promoting High-Sensitivity nAChRs in the Ventral Tegmental Area. eNeuro. 2020 Aug 12;7(4):ENEURO.0172-20.2020. doi: 10.1523/ENEURO.0172-20.2020. (Medline PMID: 32747456)

Avelar AJ, Akers AT, Baumgard ZJ, Cooper SY, Casinelli GP, Henderson BJ. Why flavored vape products may be attractive: Green apple tobacco flavor elicits reward-related behavior, upregulates nAChRs on VTA dopamine neurons, and alters midbrain dopamine and GABA neuron function. Neuropharmacology. 2019 Nov 1;158:107729. doi: 10.1016/j.neuropharm.2019.107729. Epub 2019 Jul 29. (Medline PMID: 31369741)

Henderson BJ, Grant S, Chu BW, Shahoei R, Huard SM, Saladi SSM, Tajkhorshid E, Dougherty DA, Lester HA. Menthol Stereoisomers Exhibit Different Effects on α4β2 nAChR Upregulation and Dopamine Neuron Spontaneous Firing. eNeuro. 2019 Jan 4;5(6):ENEURO.0465-18.2018. doi: 10.1523/ENEURO.0465-18.2018. (Medline PMID: 30627659)

Additional References

Mackey ED, Engle SE, Kim MR, O'Neill HC, Wageman CR, Patzlaff NE, Wang Y, Grady SR, McIntosh JM, Marks MJ, Lester HA, Drenan RM. α6* nicotinic acetylcholine receptor expression and function in a visual salience circuit. J Neurosci. 2012 Jul 25;32(30):10226-37. doi: 10.1523/JNEUROSCI.0007-12.2012. (Medline PMID: 22836257)

Nashmi R, Xiao C, Deshpande P, McKinney S, Grady SR, Whiteaker P, Huang Q, McClure-Begley T, Lindstrom JM, Labarca C, Collins AC, Marks MJ, Lester HA. Chronic nicotine cell specifically upregulates functional alpha 4* nicotinic receptors: basis for both tolerance in midbrain and enhanced long-term potentiation in perforant path. J Neurosci. 2007 Aug 1;27(31):8202-18. doi: 10.1523/JNEUROSCI.2199-07.2007. (Medline PMID: 17670967)

Henderson BJ, Srinivasan R, Nichols WA, Dilworth CN, Gutierrez DF, Mackey ED, McKinney S, Drenan RM, Richards CI, Lester HA. Nicotine exploits a COPI-mediated process for chaperone-mediated up-regulation of its receptors. J Gen Physiol. 2014 Jan;143(1):51-66. doi: 10.1085/jgp.201311102. (Medline PMID: 24378908)

Henderson BJ, Wall TR, Henley BM, Kim CH, Nichols WA, Moaddel R, Xiao C, Lester HA. Menthol Alone Upregulates Midbrain nAChRs, Alters nAChR Subtype Stoichiometry, Alters Dopamine Neuron Firing Frequency, and Prevents Nicotine Reward. J Neurosci. 2016 Mar 9;36(10):2957-74. doi: 10.1523/JNEUROSCI.4194-15.2016. (Medline PMID: 26961950)

Srinivasan R, Henley BM, Henderson BJ, Indersmitten T, Cohen BN, Kim CH, McKinney S, Deshpande P, Xiao C, Lester HA. Smoking-Relevant Nicotine Concentration Attenuates the Unfolded Protein Response in Dopaminergic Neurons. J Neurosci. 2016 Jan 6;36(1):65-79. doi: 10.1523/JNEUROSCI.2126-15.2016. (Medline PMID: 26740650)

Colony and Husbandry Information

Health Status Report

Colony Surveillance Program and Current Health Reports

Mice recovered from a cryo-archive will have health surveillance performed on recipient females. Health reports will be provided prior to shipment. If you require additional health status information, please email mmrrc@missouri.edu.

Appearance

Coat Color
Black
Eye
Black

Breeding

MMRRC Breeding System
Random intra-strain mating
Generation
F10
Overall Breeding Performance
Good

Reproductive Statistics

NOTE: "Hemizygote" as used here refers to males carrying a mutation on the X Chromosome or mice of either sex carrying an inserted transgene with no homologous allele on the other chromosome.
Viability and Fertility: Female Male Comments
Homozygotes are viable: Yes Yes
Homozygotes are fertile: Yes Yes
Hetero/Hemizygotes are fertile: N/A N/A
Age Reproductive Decline: 7 to 9 months 7 to 9 months
Average litter size
7 to 9
Recommended wean age
4 Weeks
Average Pups Weaned
7 to 9

Order Request Information

Availability Level

Limited quantities of breeder mice (recovered litter) are available from a cryoarchive; recovered litter usually available to ship in 3 to 4 months.

Cryopreserved material may be available upon request, please inquire to mmrrc@missouri.edu for more information.

A Commercial License Agreement from the Donor is required for for-profit entities to use this strain. For more information, please contact Amy J. Melton.

Conditions of Distribution

Distribution of this strain requires submission of the MMRRC Conditions of Use (COU). A link to the COU web form will be provided via email after an order has been placed; the form should be completed then or the email forwarded to your institutional official for completion.

A Commercial License Agreement from the Donor is required for for-profit entities to use this strain. For more information, please contact Amy J. Melton

Fees

Additional charges may apply for any special requests. Shipping costs are in addition to the basic distribution/resuscitation fees. Information on shipping costs and any additional charges will be provided by the supplying MMRRC facility.

Click button to Request this one strain. (Use the MMRRC Catalog Search to request more than one strain.)
MMRRC Item # Description Distribution Fee / Unit (US $)
*Shipping & Handling not included*		 Units Notes
068051-MU-SPERM Cryo-preserved spermatozoa $437.00 / $817.00
Non-Profit / For-Profit		 Aliquot Approximate quantity3
068051-MU-RESUS Litter recovered from cryo-archive $2,624.00 / $5,340.00
Non-Profit / For-Profit		 Litter Recovered litter4; additional fees for any special requests.
Cryopreserved material may be available upon request, please inquire to mmrrc@missouri.edu for more information.

1 The distribution fee covers the expense of rederiving mice from a live mouse; you will receive the resulting litter. The litter will contain at minimum one mutant carrier; the actual number of animals and the gender and genotype ratios will vary. (Typically, multiple breeder pairs can be established from the recovered litter.) Prior to shipment, the MMRRC will provide information about the animals recovered. If you anticipate or find that you need to request specific genotypes, genders or quantities of mice in excess of what is likely from a resuscitated litter, you may discuss available options and pricing with the supplying MMRRC facility.

2 An aliquot contains a sufficient number of embryos (in one or more vials or straws and based on the transfer success rate of the MMRRC facility) to transfer into one to three recipients. The MMRRC makes no guarantee concerning embryo transfer success experienced in the recipient investigator's laboratory. Neither gender nor genotype ratios are guaranteed.

3 An aliquot is one straw or vial with sufficient sperm to recover at least one litter of mice, as per provided protocols, when performed at the MMRRC facility. The MMRRC makes no guarantee concerning the success of these procedures when performed outside the MMRRC facilities.

4 The distribution fee covers the expense of resuscitating mice from the cryo-archive; you will receive the resulting litter. The litter will contain at minimum one mutant carrier; the actual number of animals and the gender and genotype ratios will vary. (Typically, multiple breeder pairs can be established from the recovered litter.) Prior to shipment, the MMRRC will provide information about the animals recovered. If you anticipate or find that you need to request specific genotypes, genders or quantities of mice in excess of what is likely from a resuscitated litter, you may discuss available options and pricing with the supplying MMRRC facility.

To request material from the MMRRC: Please fill out our on-line request form (accessible from the catalog search results page, or click the Request this Strain button in the fees section). If you have questions or need assistance completing this form, you may call Customer Service at (800) 910-2291 (in USA or Canada) or (530) 757-5710 (international calls). Before you call, please have with you: the MMRRC item number, quantity needed, Bill-to and Ship-to contact information.