STOCK Ddx25^{tm2.1(DDX25*R242H)Mld}/J

Stock number: 037675
Product name: GRTH-KI^h
Research areas: Reproductive Biology Research, Research Tools

Description

GRTH knock-in/knock-out mice have a floxed human DEAD box polypeptide 25 (DDX25) cDNA encoding the human R242H mutation replacing exons 1-5 of the mouse Ddx25 gene. Before exposure to Cre recombinase, these mice express human DDX25 R242H mutation instead of endogenous Ddx25. Upon exposure to Cre recombinase, this is designed to be a knock-out allele. These mice may be useful for studying the role of Ddx25 in spermiogenesis and male fertility.

Detailed description

DEAD (Asp-Glu-Ala-Asp) box polypeptide 25 (DDX25), also known as gonadotropin-regulated testicular RNA helicase (GRTH), is a testes-specific posttranscriptional regulator of genes that are essential for spermatid elongation and completion of spermiogenesis. GRTH knock-in/knock-out mice were generated by replacing endogenous Grth exons 1-5 with human GRTH cDNA carrying the R242H mutation. This point mutation results in ablation of phosphorylated GRTH that is found exclusively in the cytoplasm of the germ cells; this phosphoprotein is believed to shuttle mRNA between chromatoid bodies and polyribosomes. The targeting construct also includes an EGFP reporter sequence and loxP sites flanking the mutant GRTH cDNA. When bred to mice that express tissue-specific Cre recombinase, resulting offspring will have the GRTH R242H knock-in deleted in the Cre-expressing tissues. The floxed GRTH-KI^h homozygous mice are infertile as they lack sperm due to failure of round spermatid elongation during spermiogenesis. To date (September 2022), this floxed allele has not been crossed to a cre-recombinase line to produce the knock-out.

The donating investigator has made multiple Ddx25 mouse lines available: GRTH^-/- (Stock No. 024751), GRTH 6.4Kb-Tg (Stock No. 024752), GRTH 6.4Kb/del-Tg (Stock No. 024753) and GRTH-KI^h (Stock No. 037675).

Development

The GRTH knock-in/knock-out allele was generated by inserting a targeting cassette into exons 1-5 of the mouse Ddx25 gene. The targeting vector contains exon 1 of the mouse Ddx25 gene, followed by an EGFP reporter sequence, loxP site, 2A self-cleaving peptide sequence, cDNA sequence from the human GRTH gene with the R242H mutation, a human SV40 sequence, a second loxP site, an FRT-flanked neomycin selection cassette (PGK-Neo) and a third loxP site in intron 5. The construct was electroporated into 129X1/SvJ-derived GSI-1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 recipient blastocysts and the resulting chimeric males were bred to C57BL/6J females to produce heterozygous mice carrying the desired recombinant allele. Resulting progeny were then crossed to B6.Cg-Tg(Pgk1-flpo)10Sykr/J mice (Stock No. 011065) to remove the neo cassette. The resulting colony of GRTH-KI^h mice, with the neo cassette removed and the floxed human DDX25 intact, were maintained by intercrossing heterozygous mice. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J (Stock No. 000664) oocytes.

Allele

Symbol: Ddx25^{tm2.1(DDX25*R242H)Mld}
Name: targeted mutation 2.1, Maria L Dufau
MGI accession ID: MGI:6759011
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inserted expressed sequence; Humanized sequence
Marker symbol: Ddx25
Marker name: DEAD (Asp-Glu-Ala-Asp) box polypeptide 25
Chromosome: 9
Strain of origin: 129/SvJ
Molecular note: A cassette consisting of mouse exon 1, EGFP reporter sequence, loxP site, 2A self-cleaving peptide sequence, human DDX25 cDNA containing an arginine to histidine substitution at amino acid 242, SV40 sequence, loxP site, FRT-flanked PGK-neomycin selection cassette, and third loxP site replaced exons 1-5 of the gene. Flp-mediated recombination removed the neomycin cassette. The R242H variant is found in Japanese infertile men.