FLAG-Ube3a BAC transgenic mice express functional, FLAG-tagged Ube3a protein in addition to endogenous Ube3a. This increased Ube3a expression models maternal 15q11-13 duplication (dup15) and triplication (isodicentric extranumerary chromosome, idic15) that is associated with 1-3% of cases of human autism spectrum disorder. On the FVB/NJ genetic background, transgenic mice exhibit three core autism-related behavior traits (defective social interaction, impaired communication and increased repetitive stereotypies). FLAG-Ube3a BAC transgenic mice are available on a FVB/NJ genetic background (Stock No. 019730) and a C57BL/6N genetic background (Stock No. 025611).
Matthew P Anderson, Beth Israel Deaconess Medical Center / Harvard Medical School
The FLAG-Ube3a BAC transgene (also called "Ube3a-isoform2,3-3XFlag: Strain L" transgene) has a 3xFLAG tag inserted in-frame into exon 12 of Ube3a on the BAC; this generates full-length, C-terminal FLAG-tagged Ube3a mRNA long isoforms (isoforms 2 and 3, L) that are subsequently translated into functional, FLAG-tagged Ube3a protein. Transgenic mice from founder line Fd1 (FLAG-Ube3a BAC Fd1 or FLAG-Ube3a BAC X0) are described here. The FLAG-Ube3a expression pattern matched that of endogenous Ube3a across multiple brain areas (including cortex, hippocampus and thalamus). Importantly, the endogenous Ube3a gene is expressed only from the maternal chromosome in neurons; a result of paternal imprinting (the antisense transcript functions to silence paternal expression in brain). Because the FLAG-Ube3a BAC transgene lacks the transcription initiation site of the antisense transcript, expression of FLAG-Ube3a is independent of parent-of-origin or sex of the animal. Compared to wildtype animals, expression levels of Ube3a protein in whole-brain lysates are two-fold greater in Ube3a 1xTg (i.e., hemizygous) mice and three-fold greater in Ube3a 2xTg (i.e., homozygous) mice.
This increased Ube3a gene dosage models maternal 15q11-13 duplication (dup15) and triplication (isodicentric extranumerary chromosome, idic15) that is associated with 1-3% of human autism spectrum disorder. By 16 weeks of age, homozygous mice display strong penetrance of three core autism-related behavior traits; defective social interaction, impaired communication and increased repetitive stereotypies (defective three chamber social interaction task, impaired social paired vocalizations in adults and increased repetitive self-grooming, respectively). Homozygous animals exhibit reduced glutamatergic (excitatory) synaptic transmission, but no defects in GABAergic (inhibitory) synaptic transmission. To date, homozygous mice have not displayed other major confounding traits or co-morbidities (no evidence of anxiety, motor behavior deficits, memory deficits or sensory behavior deficits). Hemizygous animals are viable and fertile, with a milder phenotype compared to homozygotes. The donating investigator does not recommend breeding homozygous animals as it could result in some heretofore unknown changes in phenotype.
The FLAG-Ube3a BAC transgene (also called "Ube3a-isoform2,3-3XFlag: Strain L" transgene) was designed and generated in the laboratory of Dr. Matthew P. Anderson (Beth Israel Deaconess Medical Center / Harvard Medical School) to have a 3xFLAG tag followed by two stop codons inserted in-frame into exon 12 of Ube3a on the BAC. Specific details are below.
The ~162 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) clone RP24-178G7, containing the entire ubiquitin protein ligase E3A locus (Ube3a) as well as ~63 kbp of 5' flanking sequence and ~21 kbp of 3' flanking sequence, was obtained. DNA sequences encoding three FLAG epitope tags and two translational stop codons (DYKDHDG::DYKDHDI::DYKDDDDK::STOP::STOP) were inserted in-frame at the 3' coding/untranslated boundary of exon 12 of the Ube3a gene on the BAC via homologous recombination/BAC recombineering. A single frt site remains just downstream of the 3xFLAG region (a remnant of the frt-flanked kanamycin resistance cassette that was removed by a FLP-expressing plasmid during the BAC recombineering). This ~162 kbp FLAG-Ube3a BAC transgene was purified and then microinjected into FVB/NJ embryos. Transgenic founder animals were bred to FVB/NJ, and two founder lines with independent integration sites were established; lines Fd1 [X0] and Fd2 [X9]. The donating investigator reports that both founder lines showed the same expression pattern in the tissues examined (although line Fd2 may have slightly lower levels). Animals from line Fd1 were more extensively characterized. Line Fd1 mice were subsequently bred to produce single copy transgenic (Ube3a 1xTg [i.e., hemizygous]) animals. Hemizygous mice were bred to FVB/NJ wildtype mice for approximately 15-20 generations prior to sending albino males to The Jackson Laboratory Repository in 2014. Upon arrival, sperm was cryopreserved. To establish our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from FVB/NJ inbred females (Stock No. 001800).
Of note, SNP testing for the C57BL/6NJ-congenic FLAG-Ube3a BAC transgenic strain (Stock No. 025611; created by backcrossing Stock No. 019730 onto C57BL/6NJ) indicates a FLAG-Ube3a BAC transgene insertion site on chromosome 9.
|Expressed Gene||Ube3a, ubiquitin protein ligase E3A, mouse, laboratory|
|Expressed Gene||FLAG, FLAG octapeptide,|
|Site of Expression|
|Allele Name||transgene insertion 1, Matthew Anderson|
|Allele Type||Transgenic (Inserted expressed sequence)|
|Allele Synonym(s)||Tg(Ube3a)1Mpan; transgene insertion 1, Matthew Anderson|
|Gene Symbol and Name||Tg(Ube3a)1Mpan, transgene insertion 1, Matthew Anderson|
|Promoter||Ube3a, ubiquitin protein ligase E3A, mouse, laboratory|
|Expressed Gene||Ube3a, ubiquitin protein ligase E3A, mouse, laboratory|
|Expressed Gene||FLAG, FLAG octapeptide,|
|Strain of Origin||FVB/NJ|
|Molecular Note||The bacterial artificial chromosome (BAC) clone RP24-178G7 contains the entire ubiquitin protein ligase E3A locus (Ube3a) as well as ~63 kbp of 5' flanking sequence and ~21 kbp of 3' flanking sequence. DNA sequences encoding three FLAG epitope tags and two translational stop codons were inserted in-frame at the 3' coding/untranslated boundary of exon 12 of the gene on the BAC via homologous recombination/BAC recombineering. A single frt site remains just downstream of the 3xFLAG region. Two founder lines with independent integration sites were established; lines 1 and 2. Both founder lines exhibit the same expression pattern in the tissues examined (although line 2 may have slightly lower levels). Animals from line 1 were more extensively characterized. Ube3a expression was confirmed by Western blot of cortical lysates.|
When maintaining our live colony, hemizygous mice may be bred to wildtype (noncarrier) mice from the colony or to FVB/NJ inbred mice (Stock No. 001800). The donating investigator reports that hemizygous (Ube3a 1xTg) mice of both sexes are viable and breed perfectly well. The donating investigator does not recommend breeding homozygous (Ube3a 2xTg) animals as it could result in some heretofore unknown changes in phenotype.
When using the FLAG-Ube3a BAC transgenic on FVB/NJ mouse strain in a publication, please cite the originating article(s) and include JAX stock #019730 in your Materials and Methods section.
|Hemizygous or non carrier for Tg(Ube3a)1Mpan|
We will fulfill your order by providing at least two carriers for each strain ordered. The total number, sex, and genotypes provided will vary, although typically 8 or more animals are provided. Please check genotypes which will be recovered. While the genotypes of all animals produced will be communicated to you prior to scheduling shipment, the genotypes of animals provided may not reflect the mating scheme and genotypes described in the strain description. Animals are typically ready to ship in 11-14 weeks. If a second recovery is required to produce the minimum number of animals, then delivery time would increase to approximately 25 weeks. If we fail to produce animals of the correct genotype, you will not be charged. We cannot guarantee the reproductive success of mice shipped to your facility. If the mice are lost after the first three days (post-arrival) or do not produce progeny at your facility, a new order and fee will be necessary.
Cryorecovery to establish a Dedicated Supply for greater quantities of mice. Mice recovered can be used to establish a dedicated colony to contractually supply you mice according to your requirements. Price by quotation.
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