B6;129-Tg(tetO-Ctsb/RB1)14Smrs/J

Stock number: 032011
Product name: DN-CBRb line 14 (TetO-DN-CB-myc6-Rb1 line 14)
Research areas: Apoptosis Research, Cancer Research, Cell Biology Research, Neurobiology Research, Research Tools

Description

DN-CBRb line 14 mice have the TetO-DN-CB-myc6-Rb1 transgene that allows Tet-controllable expression of DN-CBRb - a dominant-negative, mouse lysosomal protease pre-procathepsin B (with six myc epitope tags) fused to a C-terminal truncated mutant human retinoblastoma 1. The DN-CBRb fusion is designed to exert its dominant-negative effect (proteolytic degradation) upon the endogenous RB1 protein, as well as any other proteins that associate/interact/complex with RB1. These mice may be useful for studying regulation of cell proliferation, differentiation and apoptosis.

Detailed description

Retinoblastoma 1 (RB1) has well-established roles in signaling pathways in a wide variety of tissues for the regulation of cell proliferation, differentiation and apoptosis. Complete germline or conditional RB1 deletion leads to abnormal cell proliferation followed by massive apoptosis - which may impede the study of RB1 biochemical activity.
To address this limitation, DN-CBRb line 14 mice have the TetO-DN-CB-myc6-Rb1 transgene that allows Tet-controllable expression of DN-CBRb - a dominant-negative, mouse lysosomal protease pre-procathepsin B (with six myc epitope tags) fused to a C-terminal truncated mutant human retinoblastoma 1. Once expressed, the DN-CBRb fusion is designed such that the CB cassette exerts its dominant-negative effect (proteolytic degradation) upon the endogenous RB1 protein, as well as any other proteins that associate/interact/complex with RB1.

When DN-CBRb transgenic mice are bred with another mouse expressing tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), the resulting DN-CBRb expression can be regulated with tetracycline or its analog doxycycline (dox). Cells expressing both DN-CBRb and tTA[-dox] (or rtTA[+dox]) should exhibit downregulation of RB1 expression.
For example, when bred to have the ROSA-CAG-rtTA allele (Stock No. 006965), the resulting DN-CBRb⁺/ROSA-CAG-rtTA⁺ mice show no DN-CBRb expression prior to dox exposure (no significant changes in RB1 protein expression). Following 10 days of dox-treated drinking water, DN-CBRb⁺/ROSA-CAG-rtTA⁺ mice exhibit significantly downregulated RB1 - the expected effect of DN-CBRb expression. Specifically, those DN-CBRb⁺/ROSA-CAG-rtTA⁺ [+dox] mice show inhibition of the endogenous RB1 protein in the inner ear, and in a number of other organs where RB1 is expressed [Tarang et al. 2015 Front Cell Neurosci 9:52]. In that analysis, the donating investigator used DN-CBRb line 14 hemizygous mice.

Prior to exposure to tTA (or rtTA[+dox]), hemizygous DN-CBRb line 14 mice are viable and fertile with no spontaneous behavioral or developmental abnormalities. The donating investigator reports that homozygotes are infertile, with no spontaneous behavioral or developmental abnormalities. Their DN-CBRb line 14 mice exhibit agouti, black or light brown coat color.

Development

DN-CBRb line 14 was created by Dr. Sonia M. Rocha-Sanchez (Creighton University School of Dentistry) to have Tet-controllable expression of DN-CBRb (CB-myc6-RBf1ΔC). Specifically, the donating investigator reports the TetO-DN-CB-myc6-Rb1 transgene has (from 5' to 3') the Tet response element [TRE; composed of direct repeats of the tet operator sequence [tetO]) and minimal human cytomegalovirus promoter sequences (all from pTet-Splice), a 1012 bp cDNA segment from the mouse lysosomal protease pre-procathepsin B gene (Ctsb or CB; bases 35-1047; encoding a 337 amino acid protein), six copies of Myc tag sequence (Myc6), a 1583 bp cDNA segment from the human retinoblastoma 1 gene (RB1; bases 1243-2826; amino acids 369-896; encoding a 528 amino acid, C-terminal truncated mutant RB1), and an SV40 intron and polyA sequence. A loxP-flanked ampicillin cassette was originally present downstream of the SV40 polyA sequences, but was permanently removed by a Cre recombinase vector during the in vitro assembly of the final transgene. The final ~3.4 kbp transgene was microinjected into B6129;C57BL/6 mouse zygotes. Founder mice were bred to C57BL/6J-congenic ROSA-CAG-rtTA mice (derived from Stock No. 006965) for germline transmission and simultaneous assessment of DN-CBRb expression. Mice from founder line 14 were chosen for further characterization. The DN-CBRb line 14 colony was then maintained by breeding transgenic mice with wildtype (noncarrier) mice from the colony for several generations (and the rtTA-expressing allele was removed). In 2019, DN-CBRb line 14 males (on a B6129;C57BL/6 genetic background) were sent to The Jackson Laboratory Repository. 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 DN-CBRb colony was then maintained by breeding transgenic mice together, to wildtype (noncarrier) mice from the colony or to C57BL/6J inbred mice.

Of note, FISH analyses performed by the donating investigator suggest several copies of the transgene may be inserted within chromosome 10 segment C3~D2. However, the exact transgene copy number has not been established to date (June 2018).

Allele

Symbol: Tg(tetO-Ctsb/RB1)14Smrs
Name: transgene insertion 14, Sonia M Rocha-Sanchez
MGI accession ID: MGI:5911114
Generation method: Transgenic
Attributes: Dominant negative; Inserted expressed sequence; Humanized sequence; Epitope tag
Synonyms: DN-CBRb
Marker symbol: Tg(tetO-Ctsb/RB1)14Smrs
Marker name: transgene insertion 14, Sonia M Rocha-Sanchez
Chromosome: 10
Strain of origin: 129 and C57BL/6
Expressed genes: RB1,RB transcriptional corepressor 1,human; Ctsb,cathepsin B,mouse, laboratory
Site of expression: When DN-CBRb transgenic mice are bred with another mouse expressing tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), the resulting DN-CBRb expression can be regulated with tetracycline or its analog doxycycline (dox). Cells expressing both DN-CBRb and tTA[-dox] (or rtTA[+dox]) should exhibit downregulation of RB1 expression.
Molecular note: A 1583 bp segement of the human RB1 cDNA (bases 1243-2826, amino-acids 369-896) was cloned into the pCS2+CB-Myc6 vector. This vector contains a 1012 bp (337 amino-acids) long construct of bp 35-1047 of Ctsb with 6x Myc tags fused to its C-terminus. The resulting fusion product is an 865 aa peptide comprising Ctsb, six Myc tags and Rb1. The fusion gene was then cloned into a pTet-splice vector which brings the fusion gene under the control of the tetO promoter and terminates the gene with an SV40 intron and polyA signals. This transgenic construct was used to generate several founder mice, of which this line was used for further study. The transgene results in a tetracycline-inducibele dominant negative allele that disrupts the function of Rb1-containing multimeric complexes by shuttling them to the lysosome for degradation.