STOCK Ndnf^{tm1.1(cre)Rudy}/J

Stock number: 030757
Product name: NDNF-ires-Cre knock-in , NDNF-Cre
Research areas: Neurobiology Research, Research Tools

Description

NDNF-ires-Cre knock-in mice (also called NDNF-Cre) are designed to have the endogenous neuron-derived neurotrophic factor promoter/enhancer driving Cre expression, as well as to retain endogenous Ndnf expression. These mice may be used to generate conditional mutations for studying gain-or-loss of function, fate mapping and/or circuit dissection of NDNF+ cell populations.

Detailed description

The neuron-derived neurotrophic factor protein encoded by the Ndnf gene is expressed in scattered neurons in the cerebral cortex, such as cortical interneurons. Because NDNF is a very selective marker for neocortical layer 1 interneurons (L1 INs), it may be useful for circuit dissection. NDNF is also proposed to function as a modulator that promotes endothelial cell function.

The NDNF-ires-Cre knock-in allele (also called NDNF-Cre) has an IRES sequence and Cre recombinase gene inserted after the STOP codon of the Ndnf locus. This is designed to have the endogenous Ndnf promoter/enhancer driving Cre expression, as well as to retain endogenous Ndnf expression. Specific details are below.

Mice heterozygous or homozygous for this NDNF-ires-Cre knock-in allele are viable and fertile with normal breeding, and no reported gross phenotypic or behavioral abnormalities. The donating investigator confirms cre mRNA expression (in situ hybridization) recapitulates that of endogenous Ndnf mRNA expression. While Ndnf mRNA expression pattern/levels (in situ hybridization) in somatosensory cortex from NDNF-ires-Cre heterozygotes showed no differences compared to wildtype, the donating investigator reports homozygous tissues were not tested to date (January 2019).

When NDNF-ires-Cre are bred with mice containing lox-flanked sequences, Cre-mediated recombination results in deletion of floxed sequences in NDNF expressing cells of the offspring.
For example, when NDNF-ires-Cre are bred to Rosa26^LSL-tdTomato (Ai14; see Stock No. 007914), the resulting NDNF-Cre;Ai14 mice show broad cumulative recombination patterns, which was proposed to be a result of widespread low-level expression of NDNF during development [Schuman et al. 2019 J Neurosci 39:125].
In addition, NDNF-ires-Cre mice are useful for AAV-DIO-based viral labeling to achieve specific labeling of adult NDNF-expressing cells. For example, when NDNF-ires-Cre are bred to NPY-hrGFP (Ai14; see Stock No. 006417), the resulting NDNF-Cre;NPY-hrGFP mice can be injected with AAV-DIO-fluorescent constructs for identifying populations of NDNF+/NPY+ (neurogliaform) and NDNF+/non-NPY (canopy cells) [Schuman et al. 2019 J Neurosci 39:125; also see cover image].

Development

The NDNF-ires-Cre knock-in allele (also called NDNF-Cre) was created in the laboratory of Dr. Bernardo Rudy (New York University) to have an IRES sequence and a Cre recombinase gene inserted into the 3'UTR immediately following the STOP codon of the neuron-derived neurotrophic factor gene (Ndnf) on chromosome 6 (~65.7 Mbp). The specific details are below.
The targeting vector contained, from 5' to 3', an internal ribosome entry site sequence (IRES; allows translation initiation in the middle of an mRNA sequence), the Cre recombinase sequence, a bovine growth hormone polyA sequence and a frt-flanked PGK-Neo-bGHpA cassette. The construct was electroporated into B6.Cg-Thy1^a-derived Bruce4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Chimeric mice were bred to C57BL/6J-congenic ROSA26::FLPe mice (Stock No. 009086) for germline removal of the PGK-Neo-bGHpA cassette. The resulting NDNF-ires-Cre mice were bred with C57BL/6J wildtype mice for at least one generation (and the ROSA26::FLPe allele was removed). NDNF-ires-Cre mice were subsequently bred together for several generations (black coat color was observed for almost all mice, although occasional mice with brown/agouti coat color were observed but discarded). In 2019, homozygous males (black coat color) were sent to The Jackson Laboratory Repository. See SNP results below. Upon arrival, males are used to cryopreserve sperm. To establish our live NDNF-ires-Cre colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
Of note, it is not known if the Y chromosome has been fixed to the C57BL/6J background.

In 2019, 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. This revealed 4 of 43 markers were not fixed for C57BL/6 allele-type (1 each on 4 different chromosomes). In addition, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. Collectively, these data suggest that the mice sent to The Jackson Laboratory Repository were predominantly C57BL/6 but contain some unknown genetic background contributions, and the C57BL/6 genetic background contributions may be a mix of C57BL/6J;C57BL/6N. Alternatively, all 7 of the segregating markers are C3H allele-type, which could suggest a mixed C57BL/6;C3H genetic background. Also, the SNP marker within the Thy1 was C57BL/6 allele-type (Thy1^b; Thy1.2).

Allele

Symbol: Ndnf^{tm1.1(cre)Rudy}
Name: targeted mutation 1.1, Bernardo Rudy
MGI accession ID: MGI:6284835
Generation method: Targeted
Attributes: Recombinase-expressing
Synonyms: NDNF-Cre; NDNF-ires-Cre
Marker symbol: Ndnf
Marker name: neuron-derived neurotrophic factor
Marker synonyms: A930038C07Rik; AABR07060833.1; C4orf31; epidermacan; HH25; NORD; RGD1311080
Chromosome: 6
Strain of origin: B6.Cg-Thy1^a
Site of expression: The donating investigator reports cre mRNA expression (in situ hybridization) recapitulates that of endogenous Ndnf mRNA expression. The endogenous Ndnf gene is expressed in scattered neurons in the cerebral cortex, such as cortical interneurons, and is a very selective marker for neocortical layer 1 interneurons (L1 INs).
Molecular note: The targeting vector contained, from 5' to 3', an internal ribosome entry site sequence (IRES; allows translation initiation in the middle of an mRNA sequence), the Cre recombinase sequence, a bovine growth hormone polyA sequence and a frt-flanked PGK-Neo-bGHpA cassette. Chimeric mice were bred to FLPe mice for germline removal of the PGK-Neo-bGHpA cassette.