C57BL/6-Enpp6^{em1(cre/ERT2)Lusu}/J

Stock number: 041040
Product name: Enpp6-CreER^T2
Research areas: Metabolism Research, Neurobiology Research, Research Tools

Description

Enpp6-creER^T2 knock-in mice have an IRES-cre-ERT2 fusion gene inserted immediately upstream of the 3' UTR of the Enpp6 gene. When these mice are bred with mice containing a loxP-flanked sequence of interest, tamoxifen-inducible, Cre-mediated recombination is expected to result in deletion of the floxed sequences in the Enpp6-expressing cells of the offspring. These mice may be useful for visualization and lineage tracing of pre-myelinating oligodendrocytes in the CNS during early development.

Detailed description

Ectonucleotide pyrophosphatase/phosphodiesterase 6, coded by Enpp6, is an enzyme that plays a role in choline metabolism necessary for oligodendrocyte development and myelin production in the central nervous system (CNS). ENPP6 is highly expressed in pre-myelinating oligodendrocytes during early development. Enpp6-CreER^T2 knock-in (KI) mice have an IRES-cre/ERT2 fusion sequence inserted into the 3' UTR, while retaining endogenous Enpp6 expression. This insertion is designed to have the endogenous promoter/enhancer elements direct cre/ERT2 expression to ENPP6-positive cells. When bred with mice containing a loxP-flanked sequence of interest, tamoxifen-inducible, Cre-mediated recombination will result in deletion of the floxed sequences in the ENPP6-expressing cells of the offspring.

Homozygous mice are viable and fertile with no overt phenotype. The donating investigator reports Cre/ERT2 recombinase expression/activity in ~10-20% of the ENPP6-expressing pre-myelinating oligodendrocytes in the CNS. The donating investigator has not tested the mice to knock out a floxed allele and recommends proper testing when using it for that purpose. The investigator observed specific expression in all CNS tissues, including the optic nerve and spinal cord. The donating investigator did not examine Cre/ERT2 expression in tissues other than the brain, optic nerve and spinal cord.

Development

The Enpp6-CreER^T2 knock-in allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing. A single guide RNA (5'-AGTGCTTTGGAATGAGCCAT-3') was used to introduce an internal ribosome entry site (IRES) followed by a cre/ERT2 fusion gene (Cre recombinase fused to a human estrogen receptor ligand binding domain) into the 3' UTR, immediately after the endogenous stop codon of the ectonucleotide pyrophosphatase/phosphodiesterase 6 (Enpp6) gene on Chromosome 8. Enpp6 transcript Enpp6-201 (ENSMUST00000039840.15) was used as reference for exon number and guide sequences. The sgRNA, ssDNA and the Cas9 nuclease were introduced into the cytoplasm of zygotes derived from C57BL/6J (Stock No. 000664) mice with well recognized pronuclei. Correctly targeted pups were identified by PCR and sequencing. The donating investigator reported that the resulting Enpp6-CreER^T2 knock-in mice were bred to C57BL/6 mice for at least 10 generations prior to sending to The Jackson Laboratory in 2025 (see SNP results below). To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304).

In 2025, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. The 43 markers throughout the genome suggested a C57BL/6 genetic background. Of the 5 markers that determine C57BL/6J from C57BL/6N, 4 were found to be C57BL/6N while 1 was segregating c(hromosome 8 [~57.8 Mbp]). That marker is ~10 Mb away from the targeted Enpp6 locus. These data suggest that the mice sent to The Jackson Laboratory Repository were on a C57BL/6 genetic background (predominantly C57BL/6N).

Allele

Symbol: Enpp6^{em1(cre/ERT2)Lusu}
Name: endonuclease-mediated mutation 1, Lu Sun
MGI accession ID: MGI:8242756
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing; Inducible
Marker symbol: Enpp6
Marker name: ectonucleotide pyrophosphatase/phosphodiesterase 6
Marker synonyms: 4833421B01Rik; B830047L21Rik; D8Ertd514e; E-NPP 6; Npp6
Chromosome: 8
Strain of origin: C57BL/6J
Molecular note: Using CRISPR/Cas9 endonuclease-mediated genome editing, a single guide RNA (5'-AGTAAAGCCTGCTTCCAACA-3') was used to introduce an internal ribosome entry site (IRES) followed by a cre/ERT2 fusion gene (Cre recombinase fused to a human estrogen receptor ligand binding domain) into the 3' UTR, immediately after the endogenous stop codon of the ectonucleotide pyrophosphatase/phosphodiesterase 6 (Enpp6) gene.