This triple mutant strain carries a humanized ApoE knock-in mutation (sequence coding for isoform E4), a CRISPR/cas9-generated L163P point mutation of the Clasp2 (CLIP associating protein 2) gene and a CRISPR/cas9-generated R47H point mutation of the Trem2 gene. These mice may be suitable for use in studies related to Alzheimer's disease, lipoproteins, arteriosclerosis, and coronary heart disease.
Mike Sasner, The Jackson Laboratory
Genetic Background | Generation |
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|
Allele Type | Gene Symbol | Gene Name |
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Endonuclease-mediated (Humanized sequence) | Trem2 | triggering receptor expressed on myeloid cells 2 |
Allele Type | Gene Symbol | Gene Name |
---|---|---|
Targeted (Inserted expressed sequence, Humanized sequence) | Apoe | apolipoprotein E |
Allele Type | Gene Symbol | Gene Name |
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Endonuclease-mediated (Not Specified) | Clasp2 | CLIP associating protein 2 |
This triple mutant strain carries a humanized ApoE knock-in allele, in which exons 2, 3 and most of exon 4 of the mouse Apoe gene were replaced by human APOE4 gene sequence including exons 2, 3 and 4 (and some 3' UTR sequence); a CRISPR/cas9-generated L163P point mutation of the Clasp2 (CLIP associating protein 2) and a knock-in of a point mutation into mouse Trem2, triggering receptor expressed on myeloid cells 2, gene containing a R47H point mutation, with two silent mutations. The targeted Apoe gene encodes apolipoprotein E, which is important in lipoprotein metabolism and cardiovascular disease as well as Alzheimer's disease, immunoregulation and cognition. The targeted Clasp2 gene encodes a microtubule plus-end tracking protein. The targeted Trem2 gene encodes a protein that is part of a receptor signaling complex with TYRO protein tyrosine kinase binding protein, which activates macrophages and dendritic cells during immune responses. Loss-of-function variants of the TREM2 gene are associated with Alzheimer's Disease. The R47H point mutation has been shown to increase Alzheimer's disease risk. Mice that are homozygous for the Apoetm1.1(APOE*4)Adiuj and Trem2em1Adiuj alleles, and heterozygous for the Clasp2em1Adiuj allele are viable and fertile. Homozygous viability/fertility has not been tested for the Clasp2em1Adiuj allele (March 2018). As the mice are characterized, we will modify the strain description and add phenotype data.
This triple mutant line was generated by utilizing CRISPR/cas9 endonuclease mediated genome editing of the Clasp2 gene in B6(SJL)-Apoetm1.1(APOE*4)Adiuj Trem2em1Adiuj/J (available as Stock No. 028709) mice.
The double mutant B6(SJL)-Apoetm1.1(APOE*4)Adiuj Trem2em1Adiuj/J (Stock No. 028709) was generated by crossing 2 single mutant lines.
For the Apoetm1.1(APOE*4)Adiuj allele (available as Stock No. 027894): A targeting vector containing a 3' FRT flanked neo cassette was used to replace exons 2, 3 and most of exon 4 with 1.5 kb of human APOE gene sequence including exons 2, 3 and 4 (and some 3' UTR sequence). The human ApoE sequence also contains a 2 nucleotide A to T substitution in intron 3 for identification of the targeted allele by Southern analysis. The construct was electroporated into C57BL/6J derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission by crossing to C57BL/6J. Heterozygous animals were crossed subsequently to FLP recombinase expressing mice (see Stock No. 005703) to remove the FRT site flanked PGK-neo cassette. Mice that no longer contained the FRT flanked PGK-neo cassette were then backcrossed to C57BL/6J at least once to remove the FLP recombinase transgene.
For the Trem2em1Adiuj allele (available as Stock No. 027918): Plasmids encoding a single guide RNA designed to introduce a R47H point mutation, with two silent mutations, into the Trem2 gene and the cas9 nuclease were introduced into the cytoplasm of C57BL/6J-derived fertilized eggs with well recognized pronuclei. Correctly targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by sequencing and PCR and further bred to C57BL/6J (Stock No. 000664) to develop the colony.
For the Clasp2em1Adiuj allele: plasmids encoding a single guide RNA designed to introduce the L163P point mutation (CTG>CCA) into the Clasp2 gene and the cas9 nuclease were introduced into the cytoplasm of B6(SJL)-Apoetm1.1(APOE*4)Adiuj Trem2em1Adiuj/J (Stock No. 028709)-derived fertilized eggs with well recognized pronuclei. Correctly targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by sequencing and PCR and further bred to B6(SJL)-Apoetm1.1(APOE*4)Adiuj Trem2em1Adiuj/J (Stock No. 028709). Sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize B6(SJL)-Apoetm1.1(APOE*4)Adiuj Trem2em1Adiuj/J oocytes (Stock No. 028709).
Expressed Gene | APOE, apolipoprotein E, human |
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Site of Expression | |
Site of Expression |
Allele Name | endonuclease-mediated mutation 1, MODEL-AD Center |
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Allele Type | Endonuclease-mediated (Humanized sequence) |
Allele Synonym(s) | Jax R27H ki; Trem2 R47H KI |
Gene Symbol and Name | Trem2, triggering receptor expressed on myeloid cells 2 |
Gene Synonym(s) | |
Strain of Origin | C57BL/6J |
Chromosome | 17 |
Molecular Note | The allele was generated by injecting cas9 nuclease and a single guide RNA designed to introduce a R47H point mutation with two silent mutations (lysine AAG>AAA and alanine GCC>GCA) into the gene. |
Allele Name | targeted mutation 1.1, MODEL-AD Center |
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Allele Type | Targeted (Inserted expressed sequence, Humanized sequence) |
Allele Synonym(s) | APOEepsilon4 |
Gene Symbol and Name | Apoe, apolipoprotein E |
Gene Synonym(s) | |
Expressed Gene | APOE, apolipoprotein E, human |
Strain of Origin | C57BL/6J |
Chromosome | 7 |
Molecular Note | The targeting vector contains a 3' FRT flanked neo cassette and 1.5 kb of human APOE gene sequence including exons 2, 3, 4 and a portion of the 3'UTR sequence (the APOE4 isoform) which is used to replace exons 2, 3 and most of exon 4 of the endogenous sequence. The human ApoE sequence carries a 2 nucleotide A to T substitution in intron 3. Flp-mediated recombination removed the FRT-flanked neo cassette. |
Allele Name | endonuclease-mediated mutation 1, MODEL-AD Center |
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Allele Type | Endonuclease-mediated (Not Specified) |
Allele Synonym(s) | |
Gene Symbol and Name | Clasp2, CLIP associating protein 2 |
Gene Synonym(s) | |
Strain of Origin | B6(SJL)-Apoetm1.1(APOE*4)Adiuj Trem2em1Adiuj/J |
Chromosome | 9 |
Molecular Note | A plasmids encoding a single guide RNA designed to introduce the L163P point mutation (CTG to CCA) into the gene and the cas9 nuclease were introduced into the cytoplasm of fertilized eggs with well recognized pronuclei. |
When maintaining a live colony, mice homozygous for the Apoetm1.1(APOE*4)Adiuj and Trem2em1Adiuj alleles, and heterozygous for the Clasp2em1Adiuj allele may be bred.
Homozygous viability/fertility has not been tested for the Clasp2em1Adiuj allele (March 2018).
As the mice are characterized, we will modify the strain description if necessary and add data related to viability and fertility.
When using the Clasp2*L163P/APOE4/Trem2*R47H mouse strain in a publication, please cite the originating article(s) and include JAX stock #031944 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
---|---|---|
Homozygous for Apoe<tm1.1(APOE*4)Adiuj>, heterozygous for Clasp2<em1Adiuj>, and homozygous for Trem2<em1Adiuj> or homozygous for Apoe<tm1.1(APOE*4)Adiuj>, wildtype for Clasp2<em1Adiuj>, and homozygous for Trem2<em1Adiuj> |
Frozen Mouse Embryo | B6.Cg-Apoe<tm1.1(APOE*4)Adiuj> Clasp2<em1Adiuj> Trem2<em1Adi | $2595.00 |
Frozen Mouse Embryo | B6.Cg-Apoe<tm1.1(APOE*4)Adiuj> Clasp2<em1Adiuj> Trem2<em1Adi | $2595.00 |
Frozen Mouse Embryo | B6.Cg-Apoe<tm1.1(APOE*4)Adiuj> Clasp2<em1Adiuj> Trem2<em1Adi | $3373.50 |
Frozen Mouse Embryo | B6.Cg-Apoe<tm1.1(APOE*4)Adiuj> Clasp2<em1Adiuj> Trem2<em1Adi | $3373.50 |
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The Jackson Laboratory has rigorous genetic quality control and mutant gene genotyping programs to ensure the genetic background of JAX® Mice strains as well as the genotypes of strains with identified molecular mutations. JAX® Mice strains are only made available to researchers after meeting our standards. However, the phenotype of each strain may not be fully characterized and/or captured in the strain data sheets. Therefore, we cannot guarantee a strain's phenotype will meet all expectations. To ensure that JAX® Mice will meet the needs of individual research projects or when requesting a strain that is new to your research, we suggest ordering and performing tests on a small number of mice to determine suitability for your particular project. We do not guarantee breeding performance and therefore suggest that investigators order more than one breeding pair to avoid delays in their research.
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