B6(129S4)-Kiss1^{tm1.1(cre/EGFP)Rpa}/J

Stock number: 033169
Product name: Kiss^Cre (v2), Kiss^Cre:GFP (v2)

Research areas: Neurobiology Research, Reproductive Biology Research, Research Tools

Description

Kiss1^Cre:GFP (v2) knock-in/knock-out mice express cre recombinase fused to a GFP reporter in neurons expressing the Kiss1 (KiSS-1 metastasis-suppressor) gene.

Detailed description

Transient activation of neurons in the arcuate nucleus of the hypothalamus that express kisspeptin (Kiss1, KiSS-1 metastasis-suppressor) contribute to the occurrence of hot flushes. While the vast majority of patients who suffer from hot flushes are menopausal women, hot flushes can affect both sexes in response to rapid decreases in sex hormone levels, such as that which occurs in men undergoing androgen deprivation therapy for prostate cancer or after castration.

Kiss1^Cre:GFP (v2) knock-in/knock-out mice carry a cassette encoding Cre:GFP fusion protein that replaces the coding region of the first Kiss1 coding exon. GFP is expressed constitutively from the Kiss1 promoter. Immunohistochemistry is needed to visualize the fluorescence in this line.


When crossed to a conditional reporter line (Ai14, Stock No. 007914), Kiss1 neurons are faithfully labeled in the rostral periventricular area, bed nucleus of the stria terminalis, arcuate nucleus and medial amygdala, with little to no ectopic expression.

The mutation in this strain represents an improved version of the Kiss1^{tm1.1(cre/EGFP)Stei} allele (Stock No. 017701, also called Kiss1^Cre:GFP v1) which is prone to ectopic expression when crossed to conditional reporter mice, in addition to retaining low levels of Kiss1 expression. Unlike the previous allele, homozygotes of the Kiss1^Cre:GFP (v2) line are Kiss1 null and infertile. Homozygous Kiss1^Cre:GFP (v2) males have diminished testosterone levels compared to heterozygotes.

Barring genetic crosses, the Kiss1^Cre:GFP v1 line (Stock No. 017701) remains a useful tool for visualizing Kiss1 neurons for electrophysiological recordings with the GFP-fused cre-reporter transgene. No immunohistochemistry is required to visualize GFP expression in the Kiss1^Cre:GFP v1 line. Also, viral injections into adult v1 mice, as opposed genetic crosses, can be used to gain control of those Kiss1 neurons and thereby avoid developmental ectopic recombination.

Development

In brief, a Cre:GFP fusion carrying Myc 3' UTR pA, and an FRT-SvNeo-FRT cassette were introduced to the end of the first Kiss1 exon, such that the sequence between the start codon and the end of that exon is deleted. The mutation was created via homologous recombination in C57BL/6-derived embryonic stem (ES) cells. The FRT-flanked Neo gene was removed by breeding resultant heterozygous mice with animals expressing FLPo recombinase (B6.129S4-Gt(ROSA)26Sor^tm2(FLP*)Sor, Stock No. 012930). This strain was backcrossed to C57BL/6 for 13 generations by the donating laboratory.

The mutation in this strain represents an improved version of the Kiss1^{tm1.1(cre/EGFP)Stei} allele (Stock No. 017701, also called Kiss1^Cre:GFP v1), which was prone to ectopic expression when crossed to conditional reporter mice, as well as retained levels of low expression.  The original mutation was modified by:  (a) removing the nuclear localization signal from cre, (b) using a non-optimal initiation codon, (c) removing part of the N-terminal sequence of cre, (d) adding a 3' untranslated region from the Myc gene that promotes a short mRNA half-life, (e) deleting part of exon 1 that includes the signal peptide, and (f) performing the gene targeting in C57BL/6 ES cells.

Allele

Symbol: Kiss1^{tm1.1(cre/EGFP)Rpa}
Name: targeted mutation 1.1, Richard D Palmiter
MGI accession ID: MGI:6278139
Generation method: Targeted
Attributes: Recombinase-expressing; Reporter; Null/Knockout
Synonyms: Kiss1^Cre:GFP; Kiss1^Cre (v2)
Marker symbol: Kiss1
Marker name: KiSS-1 metastasis-suppressor
Marker synonyms: Eseptin; HH13; KiSS-1; kisspeptin; metastin
Chromosome: 1
Strain of origin: C57BL/6
Molecular note: The targeting construct contains a Cre:GFP fusion carrying Myc 3' UTR pA, and an FRT-SvNeo-FRT cassette introduced to the end of the first exon, such that the sequence between the start codon and the end of that exon are deleted. Flp-mediated recombination removed the FRT-flanked neo cassette. The construct removes the nuclear localization signal from cre, uses a non-optimal initiation codon, removes part of the N-terminal sequence of cre, adds a 3' untranslated region from the Myc gene that promotes a short mRNA half-life, and deletes part of exon 1 that includes the signal peptide.