|
Allele Symbol: Chrna4tm2Lst Name: cholinergic receptor, nicotinic, alpha polypeptide 4 / targeted mutation 2, Henry A Lester Alteration at locus: Knock-In |
Genetic Alterations:
129/SvJ α4
genomic clone containing exon 5 with the L9’S mutation in the vector pKO
Scrambler V907. A neomycin resistance cassette with a phosphoglycerate
kinase promoter and polyadenylation signal flanked by loxP sites is
present 163 bp downstream from exon 5 for positive selection. The
diphtheria toxin A chain gene with an RNA polymerase II promoter has also
been added to provide negative selection for random insertion.
A 4.1 Kb
cassette containing exon 5 was removed from the pKO vector via digestion
with Spe I and inserted into the Bluescript vector for modification. PCR
mutagenesis using the QuikChange Site-directed Mutagenesis Kit was
utilized to replace the Leu9’Ser mutation with the Leu9’Ala mutation
(codon changed from
1
TCT to GCT). The presence of the mutation was
confirmed by automated sequence analysis. A small 250 bp cassette
containing the Leu9’Ala mutation was subsequently cut out from Bluescript
with BsrGI and BglII, then unidirectionally ligated into Bluescript Leu9’
Ser α4 exon 5. This was done to avoid sequencing the entire 4.1 Kb
exon 5 cassette that went through PCR amplification. The 4.1 exon 5
cassette was then removed by digestion with Spe I and ligated back into
the knock-in construct. Clones in the correct orientation yielded two
fragments, 8 and 7 Kb in length, upon digestion with Nsi I (as opposed to
10 and 5 Kb fragments in the wrong orientation). The α4 Leu9’Ala
knock-in construct was linearized with Not I and chloroform/phenol
extracted in preparation for introduction into mouse embryonic stem cells
(ES cells).
Genotype Determination:
ES Cell Line: 129/SvJ
Phenotype:
Homozygous phenotype: Viable and Fertile
Homozygous
Behavioural hypersensitivity to Nicotinic agonists
Heterozygous phenotype: Viable, fertile, anxious and hypersensitive to Nicotine induced seizers.
Check for additional phenotype data at MMRRC UC Davis
Strain Type:
Founder genetic background: 129/ SvJ
Strain genetic background: C57BL/6
Strain Development:
Donor took a complementary approach
to the KO strategy by replacing an endogenous
exon with one containing a
single point
mutation, Leu9' Y Ala9' (Leu9'Ala), within
the putative
pore-forming M2 domain of the
a4 subunit, rendering a4* receptors
hypersensitive
to nicotine.
Donor: Henry A. Lester, Ph.D., CALTECH
Primary Reference:
Tapper AR, McKinney SL, Nashmi R, Schwarz J, Deshpande P, Labarca C, Whiteaker P, Marks MJ, Collins AC, Lester HA. Nicotine activation of alpha4* receptors: sufficient for reward, tolerance, and sensitization. Science. 2004 Nov 5;306(5698):1029-32. (Medline PMID: 15528443)
Fonck C, Cohen BN, Nashmi R, Whiteaker P, Wagenaar DA, Rodrigues-Pinguet N, Deshpande P, McKinney S, Kwoh S, Munoz J, Labarca C, Collins AC, Marks MJ, Lester HA. Novel seizure phenotype and sleep disruptions in knock-in mice with hypersensitive alpha 4* nicotinic receptors. J Neurosci. 2005 Dec 7;25(49):11396-411. (Medline PMID: 16339034)
None
Mice recovered from a cryo-archive will have health surveillance performed on the
resuscitated animals.
Limited quantities of breeder mice (recovered litter) are available from a cryoarchive; recovered litter usually available to ship in 3 to 4 months.
* 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 $) |
Units |
|---|---|---|---|
| 029171-UCD-Resus | Resuscitation from cryopreserved material | $1,850.00 | litter a |
| 029171-UCD-Sperm | Cryopreserved spermatozoa | $400.00 | aliquot b |
| 029171-UCD-Embryos | Cryopreserved embryos | $950.00 | aliquot c |
a 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 breedable 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 repository 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 repository.
b An aliquot contains sufficient material (volume and concentration) for at least two IVF or several artificial inseminations (based on our procedures). The MMRRC makes no guarantee concerning success of these procedures when performed outside the MMRRC facilities.
c An aliquot contains a sufficient number of embryos (in one or more vials and based on the transfer success rate of the MMRRC facility) to transfer to at least two recipients. The MMRRC makes no guarantee concerning embryo transfer success experienced in the recipient investigator's laboratory. Neither gender nor genotype ratios are guaranteed.
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 above). For additional information about this strain, please email Customer Service at service@mmrrc.org or call (800) 910-2291 (in USA or Canada) or (207) 288-6009 (international calls).
MMRRC Contact Information
Home Page
The MMRRC is a collaborative effort, funded by grants from the National Center for Research Resources,
NIH, DHHS.
Web forms on this site assume JavaScript enabled for Netscape 6.x, Microsoft Internet Explorer 5.5 and later versions; forms will not function as expected with earlier versions or when JavaScript is turned off.
Last Modified: April 29, 2008