C57BL/6J-Stmn2^{em8(STMN2*)Lutzy}/Mmjax

Stock number: 035721
Product name: Stmn2^(HumΔGU)
Research areas: Cell Biology Research, Mouse/Human Gene Homologs, Neurobiology Research

Description

Stmn2^em8(STMN2*) is a CRISPR/cas9 generated mutant of the stathmin-like 2 (Stmn2) gene carrying 222 nt of human STMN2 exon 2a. The STMN2 sequence is modified to include the human MS2 stem loop sequence and replace the TDP43 binding element. These mice may be useful in pre-clinical studies of Amyotrophic Lateral Sclerosis (ALS).

Detailed description

Stmn1 (stathmin-like 2) encodes a phosphoprotein that is involved in microtubule dynamics and signal transduction. Specifically, it is involved in the regulation of neuronal growth. Stmn2^em8(STMN2*) knockin mice were created using CRISPR/cas9 genome editing to insert 222 nt of the human STMN2 exon 2a and flanking regions into intron 1 of the mouse Stmn2 gene. The STMN2 sequence is modified to include the human MS2 stem loop sequence and replace the TDP43 binding element. The targeted Stmn2 gene encodes a protein involved in microtubule stability. Mice heterozygous for the mutation are viable and fertile. There are fewer homozygotes (11.8%) produced from a heterozygote x heterozygote mating than expected, suggesting a subviable homozygous phenotype with either embryonic or early perinatal loss. As the mice are characterized, we will modify the strain description and add phenotype data.

Development

The Stmn2^em8(STMN2*) allele was generated using CRISPR/cas9 endonuclease-mediated genome editing. Guide RNAs were selected to target intron 1 of the stathmin-like 2 gene (Stmn2) and insert a modified human SMNT2 exon 2a. The humanized/mutant exon 2a in this strain is 222nt in length and is flanked by 1491 bp and 1548 bp of human genomic DNA, located 5’- and 3’- of exon 2a, respectively.  The 19-nt MS2 stem loop contains the sequence 5’-ACATGAGGATCACCCATGT-3’ was used to replace the 24-nt TDP-43 binding sequence 5’-TGTGTGAGCATGTGTGCGTGTGTG-3’ in the 3’-UTR of the exon2a-containing mouse-human hybrid STMN2 prematurely terminated mRNA transcript. The donor plasmid, both guides and cas9 nuclease were introduced into single cell C57BL/6J zygotes and transferred to pseudopregnant females. Note that the flanking mouse intron 1 sequences present in the donor plasmid was derived from N2A cell line.  There are three SNPs in the left arm that differ between the C57BL/6J sequence reported in Ensembl.  It is not expected that any of these SNPs impact the usage of exon 2a. Progeny were screened by DNA sequencing to identify correctly targeted pups, which were then bred to C57BL/6J mice (Stock No. 000664) for germline transmission. N1 progeny derived from a single correctly targeted founder carrying the allele were backcrossed to C57BL/6J for at least two generations to establish the colony.

Allele

Symbol: Stmn2^{em8(STMN2*)Lutzy}
Name: endonuclease-mediated mutation 8, Catherine Lutz
MGI accession ID: MGI:7343684
Generation method: Endonuclease-mediated
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Stmn2
Marker name: stathmin-like 2
Chromosome: 3
Strain of origin: C57BL/6J
Molecular note: CRISPR/cas9 endonuclease-mediated genome editing uses guide RNAs were selected to target intron 1 and insert a modified human SMNT2 exon 2a. The humanized/mutant exon 2a in this strain is 222nt in length and is flanked by 1491 bp and 1548 bp of human genomic DNA, located 5'- and 3'- of exon 2a, respectively. The 19-nt MS2 stem loop contains the sequence 5'-ACATGAGGATCACCCATGT-3' is used to replace the 24-nt TDP-43 binding sequence 5';-TGTGTGAGCATGTGTGCGTGTGTG-3' in the 3';-UTR of the exon2a-containing mouse-human hybrid STMN2 prematurely terminated mRNA transcript.