B6;D2-Tn(pb-CAG-dcas9,-EGFP)1Yangh/J

Stock number: 031645
Product name: Cre-dependent SPH (SunTag-p65-HSF1)
Strain synonyms: CRISPR-dCas9-activator transgenic mice, B6;D2-Tn(pb-CAG-cas9*,-EGFP)1Yangh/J
Research areas: Neurobiology Research, Research Tools

Description

SPH mice express dCas9 (nuclease-deactivated) and EGFP in a Cre recombinase inducible system to allow simultaneous overexpression of multiple genes and lncRNAs, when an sgRNA array is used. When used in combination with single guide RNAs and a Cre source, these mice have applications in studies of mRNA dosing effects, in vivo genome-wide gain-of-function screens, disease modeling and in vivo cell reprogramming.

See Detailed Description section for updated information regarding discovery of the transgene insertion site on chromosome 14 within Dpysl2 intron 3 with no transgene integration detected near Gm33925 on chromosome 3 [July 2022].

Detailed description

These SPH mice have Cre recombinase-inducible expression of nuclease-deactivated dCas9 and EGFP, under the control of the of the CAG promoter. When crossed to a strain expressing Cre recombinase, or transfected with cre-expression plasmids to remove floxed STOP cassette, the resulting progeny mice will express both dCas9 and EGFP in the cre-expressing tissues. Induction of dCas9 can also be detected using antibodies (see below). Hemizygotes are viable and fertile. Homozygous viability/fertility has not been tested (as no genotyping assay to distinguish hemizygous from homozygous was developed [April 2018; October 2020]).

When used in combination with single guide RNAs and a Cre source, they allow simultaneous activation and overexpression of multiple mouse genes and long noncoding RNAs in vivo or ex vivo. In 2018, the donating investigator reported that inverse PCR analysis showed a transgene insertion site downstream of the Gm33925 locus [PMID:29335603); Supplementary Figure 4a], but also that other undetected insertion sites are possible. See further below for updated information regarding discovery of the transgene insertion site on chromosome 14 within Dpysl2 intron 3 with no transgene integration detected near Gm33925 on chromosome 3 [July 2022].

When crossed to mice expressing Cre recombinase in GFAP-Cre (Stock No. 004600), transgene expression is detected in astrocytes. When sgRNAs for neurogenic transcription factors (Ascl1, Neurog2 and Neurod1) were delivered to the resulting bi-transgenic mice, mature astrocytes were converted into neurons.

The donating investigator reports expression is not visible by EGFP fluorescence, instead the induction of dCas9 can be detected using antibodies, specifically using primary antibody as rabbit monoclonal antibody to HA-tag (1:1000, #3724, CST) and secondary antibody as goat anti-rabbit Alexa Fluor 488 (1:1000, #A-11034, Thermo Fisher). [June 2020]

The Jackson Laboratory live colony was originally maintained by breeding hemizygous mice with wildtype from the colony (also some were bred to C57BL/6J and/or B6D2F1/J). In 2019, some hemizygous mice spontaneously began to exhibit excessive urination and high glucose. We discontinued breeding those animals. At that time, our colony maintenance plan was changed to breeding hemizygotes (normal urination/glucose) with B6D2F1/J every generation. The spontaneous urination/glucose phenotype was subsequently eliminated in our live colony. We then froze sperm in February 2020.

Transgene insertion site and copy number update [July 2022]:
 In 2022, advanced quality control analysis was concluded by The Jackson Laboratory (JAX) that Stock No. 031645 has one copy of the transgene inserted on chromosome 14 within Dpysl2 intron 3. In contrast to the donating investigator's original publication [Zhao et al., 2018 Nat Neurosci 21:440 (PMID:29335603); Supplementary Figure 4a], our analyses found no transgene integration detected near Gm33925 on chromosome 3. Specific details are below.
 JAX Nanopore analyses (2021-2022) identified one copy of the transgene is inserted on chromosome 14 ~67,089,601 [GRCm39] which corresponds to intron 3 within the dihydropyrimidinase-like 2 locus (Dpysl2 ; transcript Dpysl2-201). It is not known if the transgene insertion has any effect on Dpysl2 expression. Sequencing confirmed the transgene retained all the expected parts (including the NeoR at its 3' end), as well as the transposable element sequence at its 5' end. There is no transposable element sequence at its 3' end. Furthermore, our analyses found no transgene integration detected near Gm33925 on chromosome 3.
 The original JAX genotyping assay utilized primers within the transgene sequence (transgene-only assay). Following the Nanopore and sequencing analyses, we additionally created an assay with primers in the transgene sequence as well as in the Chr14 genomic insertion region (Chr14-specific assay). Sampling our live breeding units (2022) and the mice recovered from cryopreserved bankstock (BS31645ASP; hemizygous sperm frozen in 2020) revealed all mice hemizygous for the transgene-only assay were also hemizygous for the Chr14-specific assay, and there were no examples of mice negative for the transgene-only assay but positive for the Chr14-specific assay.
 Taken together, this indicates our live mice (2022) and cryopreserved bankstock (2020) have only the Chr14 insertion and not the Chr3 insertion. These results did not determine if Stock No. 031645 was sent to The Jackson Laboratory in 2018 having only the Chr14 insertion, or both the Chr14 and Chr3 insertions, or additional insertion sites. It is possible that our 2019-2020 efforts to breed away from a spontaneous urination phenotype could have removed the Gm33925 insertion site. It cannot be determined which insertion(s) researchers may have received from JAX before 2022. Researchers can assay their mice using our transgene-only assay and Chr14-specific assay which are linked in the Genotyping Protocols section below.

Development

A targeting vector containing a CAG promoter, floxed STOP cassette, dCas9 sequence, HA tag, 10 copies of the GCN4 (General Control Protein) epitope peptide sequence, P2A, scFv sequence encoding cognate single-chain antibody that recognizes GCN4, P65-HSF1 activator helper complex, T2A, EGFP, woodchuck hepatitis post transcriptional regulatory element (WPRE) and polyA signal, was utilized in the construction of this reporter. The donating laboratory also reports a PGK-neo cassette is present downstream of the polyA signal. This construct was flanked by piggyBac transposase inverted terminal repeats and was co-injected with a plasmid encoding piggyBac transposase expressed under the control of a beta-actin promoter into C57BL/6 X DBA/2J F1 zygotes. The resulting chimeric animals were crossed to C57BL/6J mice.
In 2018, the donating investigator reported that inverse PCR analysis showed a transgene insertion site downstream of the Gm33925 locus [PMID:29335603); Supplementary Figure 4a], but also that other undetected insertion sites are possible. See Detailed Description section updated information regarding discovery of the transgene insertion site on chromosome 14 within Dpysl2 intron 3 with no transgene integration detected near Gm33925 on chromosome 3 [July 2022].
Males were sent to The Jackson Laboratory in 2018. To establish our live colony, we originally maintained by breeding hemizygous mice with wildtype from the colony, also some were bred to C57BL/6J (Stock No. 000664) and/or B6D2F1/J (Stock No. 100006). In 2019, some hemizygous mice spontaneously began to exhibit excessive urination and high glucose. We discontinued breeding those animals. At that time, our colony maintenance plan was changed to breeding hemizygotes (normal urination/glucose) with B6D2F1/J every generation [September 2019]. The spontaneous urination/glucose phenotype was subsequently eliminated in our live colony. We then froze sperm in February 2020.

Allele

Symbol: Tn(pb-CAG-dcas9,-EGFP)1Yangh
Name: transposon insertion 1, Hui Yang
MGI accession ID: MGI:6149220
Generation method: Transposon induced
Attributes: Conditional ready (e.g. floxed); Reporter; Epitope tag; Endonuclease
Synonyms: SPH; Tn(pb-CAG-cas9*,-EGFP)1Yangh
Marker symbol: Tn(pb-CAG-dcas9,-EGFP)1Yangh
Marker name: transposon insertion 1, Hui Yang
Marker synonyms: Tn(pb-CAG-cas9*,-EGFP)1Yangh
Chromosome: 14
Strain of origin: (C57BL/6 x DBA/2J)F1
Expressed genes: cas9,CRISPR associated protein 9,; GFP,Green Fluorescent Protein,
Site of expression: When crossed to a strain expressing Cre recombinase, or transfected with cre-expression plasmids to remove floxed STOP cassette, the resulting progeny mice will express both dCas9 and EGFP in the cre-expressing tissues.
Molecular note: The targeting vector contains a CAG promoter, floxed STOP cassette, dCas9 sequence, HA tag, 10 copies of the GCN4 (General Control Protein) epitope peptide sequence, P2A, scFv sequence encoding cognate single-chain antibody that recognizes GCN4, P65-HSF1 activator helper complex, T2A, EGFP, and a woodchuck hepatitis post transcriptional regulatory element (WPRE). This construct was flanked by piggyBac transposase inverted terminal repeats and was co-injected with a plasmid encoding piggyBac transposase expressed under the control of a beta-actin promoter. Genetic analysis at The Jackson Laboratory indicates that one copy of the transgene inserted on chromosome 14 within Dpysl2 intron 3, rather than than the Gm33925 locus as originally reported. It is not known if the transgene insertion has any effect on Dpysl2 expression.