TetO-Bcl2-IRES-tdTomato transgenic mice have Tet-inducible/controllable expression of the long isoform of mouse Bcl2 and the tdTomato fluorescent protein. These mice may be useful for studying the anti-apoptotic effects of BCL2 (i.e., extended cell survival), accompanied by tdTomato fluorescence.
C. Ron Yu, Stowers Institute for Medical Research
Navigator neurons are a group of perinatally born olfactory sensory neurons that are proposed to play an essential role in establishing the olfactory map during the critical period of nervous system / neural circuit development (Wu et al. 2018 Neuron 100:1066). The anti-apoptotic B-cell lymphoma-2 family protein BCL-2 is a key regulator of programmed cell death, and therefore has important function in development, homeostasis and disease (malignancies, neurodegeneration, etc.).
tetO-Bcl2-IRES-tdTomato transgenic mice have Tet-inducible/controllable expression of the long isoform of mouse BCL2 and the tdTomato fluorescent protein. When tetO-Bcl2-IRES-tdTomato transgenic mice are bred with another mouse expressing tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), the resulting BCL2 and tdTomato expression can be regulated with tetracycline or its analog doxycycline (dox). This is designed such that cells expressing both BCL2 and tTA[-dox] (or rtTA[+dox]) should exhibit the anti-apoptotic effects of BCL2 (i.e., extended cell survival), accompanied by tdTomato fluorescence.
Prior to exposure to tTA (or rtTA[+dox]), mice hemizygous for the tetO-Bcl2-IRES-tdTomato transgene are viable and fertile with normal breeding, and no spontaneous phenotypic abnormalities. The donating investigator reports this transgene is not leaky (i.e., no tdTomato expression is observed before tTA introduction). It has not been attempted to make this strain homozygous to date (March 2019).
Breeding tetO-Bcl2-IRES-tdTomato mice to have the Omp-IRES-tTA allele (Stock No. 017754) and the tetO-tTA*-tauLacZ transgene (from Stock No. 008344) creates the triple mutant Omp-TetTag:tetO-Bcl2-IRES-tdTomato mice. Prior to dox administration, the olfactory marker protein-driven tTA results in expression of BCL2, tdTomato, lacZ and tTA*H100Y in mature olfactory sensory neurons (mOSNs). Because the tetO-tTA*-tauLacZ transgene encodes the tetracycline-insensitive tTA*H100Y, once it is initially induced by another source of tTA (e.g., Omp-IRES-tTA), expression of tTA*H100Y is sustained by a self-perpetuating loop - even in the presence of dox. Omp-TetTag:tetO-Bcl2-IRES-tdTomato mice exhibit sustained expression of BCL2 - resulting in extended survival of OSNs in the neuroepithelium and more exuberant axons in the EPL. Additionally, tdTomato fluorescence was robust/bright, and there were more lacZ+ neurons in the neuroepithelium compared to controls. [Wu et al. 2018 Neuron 100:1066]
The tetO-Bcl2-IRES-tdTomato transgene was created by Dr. C. Ron Yu (Stowers Institute) to have (from 5' to 3') the Tet response element (TRE; ~600 bp sequence composed of direct repeats of the tet operator sequence [tetO] and minimal cytomegalovirus promoter sequence [Pmin]), a ~700 bp cDNA segment encoding the long isoform of the B cell leukemia/lymphoma 2 gene (Bcl2; PCR-amplified from mouse olfactory epithelium cDNA), an internal ribosome entry site (IRES; allows translation initiation in the middle of an mRNA sequence), a tdTomato fluorescent protein (a non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein [tandem dimer]) and an SV40 polyA sequence.
The final 3864 bp transgene was microinjected into FVB mouse zygotes. Founder mice were bred to C57BL/6J for germline transmission. Mice from founder line LM1 were chosen for further characterization. The TetO-Bcl2-IRES-tdTomato line LM1 colony was then backcrossed to C57BL/6J for at least five generations prior to sending males (black coat color) to The Jackson Laboratory Repository in 2019. Upon arrival, sperm was cryopreserved. To generate the living colony, an aliquot of the frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664). The TetO-Bcl2-IRES-tdTomato colony was then maintained by breeding transgenic mice together, to wildtype (noncarrier) mice from the colony or to C57BL/6J inbred mice.
Of note, the donating investigator reports that, at least once during backcrossing, a TetO-Bcl2-IRES-tdTomato female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin). The transgene insertion site(s) and copy number are not known to date (March 2019).
|Allele Name||transgene insertion LM1, Ron Yu|
|Allele Type||Transgenic (Reporter, Inducible, Inserted expressed sequence)|
|Gene Symbol and Name||Tg(tetO-Bcl2,-tdTomato)LM1Ryu, transgene insertion LM1, Ron Yu|
|Strain of Origin||FVB/N|
|Molecular Note||The transgenic construct contains (from 5' to 3') the Tet response element (TRE; ~600 bp sequence composed of direct repeats of the tet operator sequence [tetO] and minimal cytomegalovirus promoter sequence [Pmin]), a ~700 bp cDNA segment encoding the long isoform of the B cell leukemia/lymphoma 2 gene, an internal ribosome entry site, a tdTomato fluorescent protein and an SV40 polyA sequence.|
When maintaining our live colony, transgenic carrier mice may be bred together, to wildtype (noncarrier) mice from the colony or to C57BL/6J inbred mice (Stock No. 000664).
Prior to exposure to tTA (or rtTA[+dox]), mice hemizygous for the tetO-Bcl2-IRES-tdTomato transgene are viable and fertile with normal breeding, and no spontaneous phenotypic abnormalities. It has not been attempted to make this strain homozygous to date (March 2019).
When using the tetO-Bcl2-IRES-tdTomato mouse strain in a publication, please cite the originating article(s) and include JAX stock #033125 in your Materials and Methods section.