Strain Detail Sheet

Strain Name:
C57BL/6N-Chrnb4tm1.1Geno/Mmucd
Stock Number:
065828-UCD
Citation ID:
RRID:MMRRC_065828-UCD
Other Names:
C57BL/6ChrnBeta4-GFP

Strain Information

Gene Details

Chrnb4tm1.1Geno
Name: cholinergic receptor, nicotinic, beta polypeptide 4; targeted mutation 1.1, Genoway
Type: Allele
Species: Mus musculus (mouse)
Chromosome: 9
Alteration at locus: Knock-In
Chrnb4
Name: cholinergic receptor, nicotinic, beta polypeptide 4
Synonyms: Acrb-4, Acrb4
Type: Gene
Species: Mouse
Chromosome: 9
Alteration at locus: Knock-In
NCBI: 108015
VEGA: 9
HGNC: HGNC:1964
Homologene: 20196
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Genetic Alterations
GFP was inserted in-frame into the M3-M4 loop region of the β4 nAChR subunit coding sequence. GFP insertion was executed using a modified, two-step QuikChange (Stratagene) reaction according to our previously published methods (PMID:17932221). To construct nAChRs with XFP inserted into the M3-M4 loop, a two-step PCR protocol was used. First, YFP or CFP was amplified with PCR using oligonucleotides (sequences available upon request) designed to engineer 5′ and 3′ overhangs of 15 base pairs that were identical to the site where XFP was to be inserted, in-frame, into the nAChR M3-M4 loop. A Gly-Ala-Gly flexible linker was engineered between the nAChR sequence and the sequence for YFP/CFP at both the 5′ and 3′ ends. In the second PCR step, 100 ng of the first PCR reaction was used as a primer pair in a modified QuikChange reaction using Pfu Ultra II (Stratagene, Cedar Creek, TX) polymerase and the appropriate nAChR cDNA as a template. Briefly, an Ala-Gly-Ala linker was inserted immediately 5′ and 3′ to the GFP coding sequence. AlaGlyAla-GFP-AlaGlyAla was inserted between residues Gly378 and Pro379 in mouse β4.
Genotype Determination
  • Genotyping Protocol(s)
  • Center protocol and contact for technical support will be shipped with mice.
    • ES Cell Line
    Not specified

    Strain Description

    Phenotype
    None/Normal/Wild-type
    MeSH Terms
    • Animals
    • Choline O-Acetyltransferase/metabolism
    • Dose-Response Relationship, Drug
    • Electric Stimulation
    • Excitatory Amino Acid Agents/pharmacology
    • Female
    • Gene Expression/drug effects
    • Gene Expression/genetics
    • Green Fluorescent Proteins/genetics
    • Habenula/cytology
    • Habenula/drug effects
    • Habenula/metabolism
    • In Vitro Techniques
    • Male
    • Membrane Potentials/drug effects
    • Membrane Potentials/genetics
    • Membrane Potentials/physiology
    • Mice
    • Mice, Inbred C57BL
    • Mice, Transgenic
    • Neurons/drug effects
    • Neurons/metabolism
    • Neurons/physiology
    • Nicotine/pharmacology
    • Patch-Clamp Techniques
    • Protein Subunits/genetics
    • Protein Subunits/metabolism
    • Receptors, Nicotinic/genetics
    • Receptors, Nicotinic/physiology
    • alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/pharmacology
    Strain Development
    A targeting vector was constructed containing exon 5 with GFP fused in-frame in the desired position in the M3-M4 loop coding sequence. Several kilobases of 5′ and 3′ sequence (relative to exon 5) were included to direct homologous recombination. A neomycin cassette flanked by loxP sites was inserted adjacent to the modified exon 5 in the targeting vector. A diphtheria toxin negative selection cassette was inserted adjacent to one of the homology arms (5′ or 3′). Targeting vectors were linearized and electroporated into C57BL/6 embryonic stem cells. G418-resistant cell clones were isolated and amplified. First, clones were screened via PCR to identify those positive for both the 5′ and 3′ homologous recombination events. Next, clones positive in the 5′ and 3′ PCR assays were further evaluated for correct homologous recombination (and the absence of additional genomic rearrangements) via Southern blot with probes designed to detect both the 5′ and 3′ homologous recombination events. At least three positive ES cell clones were injected into 3.5-d-old embryos (blastocyst stage) derived from an albino C57BL/6 strain (C57BL/6J-Tyrc-2J/J), and reimplanted into pseudo-pregnant females and allowed to develop to term. Progeny males with evidence of high (>50%) chimerism (based on black vs. white coat color) were selected for further breeding after reaching sexual maturity. To both (1) breed chimeras to the F1 generation and (2) excise the neomycin selection cassette from the genome of the F1 progeny, chimeric males were mated with female C57BL/6N Cre-deleter (proprietary to genOway) mice. To confirm germline transmission, neo-excision, and maintenance of the proper homologous recombination event, F1 mice were genotyped via both PCR and Southern blot. The β4-GFP mice are fully isogenic with C57BL/6.
    Suggested Control Mice
    C57BL/6N

    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@ucdavis.edu. Older strains may not have this information.

    Research Applications

    • Models for Human Disease
    • Neurobiology

    Strain Origin

    Donor
    Henry Lester, Ph.D., California Institute of Technology.
    Ryan Drenan, Ph.D., Wake Forest University, School of Medicine.
    Primary Reference

    Shih PY, Engle SE, Oh G, Deshpande P, Puskar NL, Lester HA, Drenan RM.Differential expression and function of nicotinic acetylcholine receptors insubdivisions of medial habenula. J Neurosci. 2014 Jul 16;34(29):9789-802. doi:10.1523/JNEUROSCI.0476-14.2014. (Medline PMID: 25031416)

    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@ucdavis.edu.

    Appearance

    Coat Color
    Black
    Eye
    Black

    Breeding

    MMRRC Breeding System
    Random intra-strain mating
    Generation
    N10+ (C57BL/6NCrl)
    Overall Breeding Performance
    Good

    Reproductive Statistics

    Viability and Fertility: Female Male Comments
    Homozygotes are viable: Yes Yes
    Homozygotes are fertile: Yes Yes
    Heterozygotes are fertile: Yes Yes
    Age Reproductive Decline: 4 to 6 months 10 to 12 months
    Average litter size
    4 to 6
    Recommended wean age
    3 Weeks
    Average Pups Weaned
    4 to 6

    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@ucdavis.edu for more information.

    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.

    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
    065828-UCD-SPERM Cryo-preserved spermatozoa $546.25 / $869.88
    Non-Profit / For-Profit    		 Aliquot Approximate quantity3
    065828-UCD-RESUS Litter recovered from cryo-archive $4,044.00 / $7,650.23
    Non-Profit / For-Profit    		 Litter Recovered litter4; additional fees for any special requests.
    Cryopreserved material may be available upon request, please inquire to mmrrc@ucdavis.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.