FVB/NJ-Tg(Ube3a)1Mpan/J

Stock number: 019730
Product name: FLAG-Ube3a BAC transgenic on FVB/NJ
Research areas: Developmental Biology Research, Neurobiology Research, Research Tools

Description

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).

Detailed description

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.



Of note, a floxed, non-FLAG tagged, Ube3a BAC transgenic line is available as Stock No. 036631.

Development

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.

Allele

Symbol: Tg(Ube3a)1Mpan
Name: transgene insertion 1, Matthew Anderson
MGI accession ID: MGI:5576240
Generation method: Transgenic
Attributes: Inserted expressed sequence
Marker symbol: Tg(Ube3a)1Mpan
Marker name: transgene insertion 1, Matthew Anderson
Chromosome: UN
Strain of origin: FVB/NJ
Expressed genes: Ube3a,ubiquitin protein ligase E3A,mouse, laboratory; FLAG, FLAG octapeptide,
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.