The Hand2fl allele has loxP sites flanking exon 1 (PGK-neo removed) of the Hand2 gene. These floxed mice are used to generate global or tissue-specific Hand2 deletion mice, which allow studies of this transcription factor in sympathetic nervous system morphogenesis and cell type-specific gene expression in sympathetic, enteric and adrenal chromaffin cells, cardiac morphogenesis and limb bud / branchial arch development. The absence of Hand2 expression may also be a useful biomarker for atypical hyperplasia, endometrioid carcinoma and epigenetic silencing.
Anthony B Firulli, Indiana University School of Medicine
The Hand2fl allele has loxP sites flanking exon 1 of the Hand2 gene. Because the PGK-neo cassette is not present in this allele, heterozygotes (Hand2fl/+) and homozygotes (Hand2fl/fl) are viable and fertile with no alterations in Hand2 expression levels/patterns (in situ hybridization and qRT-PCR) compared to wildtype mice. Homozygotes exhibit no reported abnormal phenotype.
Hand2 is a basic helix-loop-helix family transcription factor that functions in sympathetic nervous system morphogenesis and cell type-specific gene expression in sympathetic, enteric and adrenal chromaffin cells, cardiac morphogenesis and limb bud / branchial arch development.
The absence of Hand2 expression may also be a useful biomarker for atypical hyperplasia, endometrioid carcinoma and epigenetic silencing.
When Hand2fl mice are bred to Cre recombinase-expressing animals, the resulting offspring may be useful in generating global Hand2 knockout mice or tissue-specific Hand2 knockout mice. Specific examples are described below.
When bred to germline Cre-expressing mice, the resulting Hand2 global knockout homozygotes (Hand2ko or Hand2Delta) are embryonic lethal around embryonic day (E)10-12, with some surviving to birth but expiring thereafter.
Breeding Hand2fl mice to Wnt1-Cre transgenic mice (Stock Nos. 003829 / 009107) results in deletion of Hand2 expression in neural crest cells. The resulting Hand2cko homozygotes exhibit reduced neuronal precursor cell/neuroblast proliferation and neuronal numbers, abnormal facial/skull development and aglossia (absence of a tongue), with no gross cleft palate. Hand2cko mice also have neurogenesis defects/decreased neurons in the developing enteric nervous system with few to no noradrenergic sympathetic ganglion neurons.
Deletion of Hand2 expression in neural precursor cells using Nestin-Cre transgenic mice (Stock Nos. 003771 / 019103) is lethal at around postnatal day (P)20, allowing analysis throughout development and the early neonatal period. Such mice have abnormal enteric nervous system development with loss of all nitergic neurons, decreased numbers of cholinergic neurons and functional aganglionosis.
A targeting vector was designed by Dr. Marthe J. Howard (University of Toledo) to insert a loxP site upstream of exon 1, and a loxP-flanked PGK-neo cassette site downstream of exon 1 of the heart and neural crest derivatives expressed transcript 2 gene (Hand2) on chromosome 8. The construct was electroporated into 129S1/SvImJ-derived embryonic stem (ES) cells. Correctly targeted ES cells were were identified and injected into recipient blastocysts. The chimeric animals were bred to C57BL/6J mice to establish germline transmission. The Hand2loxpneop allele was found to be a hypomorph, so the mice were then bred to Sycp1-Cre mice (undisclosed genetic background; Stock No. 003466) for germline removal of the PGK-neo cassette. The resulting offspring that retained the loxP-flanked exon 1 with the PGK-neo removed were selected and called Hand2fl (also Hand2loxp, Hand2ploxp or Hand2fl Hand2flox). The Hand2fl colony was maintained on a mixed C57BL/6;129S genetic background and the Cre transgene was removed. In 2015, Hand2fl breeders were sent to Dr. Anthony B. Firulli (Indiana University School of Medicine). There, Hand2fl mice were bred together to make homozygous. In 2015, Dr. Firulli sent Hand2fl males on a mixed C57BL/6;129S genetic background (agouti coat colors) to The Jackson Laboratory Repository. Upon arrival, mice were backcrossed to C57BL/6J inbred mice (Stock No. 000664) to for at least two generations to establish our living Hand2fl mouse colony (negative for neo and Cre). Later, sperm was cryopreserved.
|Allele Name||targeted mutation 1.1, Marthe J Howard|
|Allele Type||Targeted (Conditional ready (e.g. floxed), No functional change)|
|Gene Symbol and Name||Hand2, heart and neural crest derivatives expressed 2|
|Strain of Origin||129S1/SvImJ|
|Molecular Note||A loxP site was inserted upstream of exon 1 and a floxed neo cassette was inserted downstream of exon 1. Germ line, cre-mediated recombination was used to remove the neo cassette leaving exon 1 floxed.|
When maintaining a live colony, heterozygous mice may be bred together, to wildtype mice from the colony or to C57BL/6J inbred mice (Stock No. 000664). Alternatively, homozygous mice may be bred together.
When using the Hand2 floxed (Hand2loxp , Hand2ploxp , Hand2fl , Hand2flox) mouse strain in a publication, please cite the originating article(s) and include JAX stock #027727 in your Materials and Methods section.