A knock-in allele of Hand2 expressing Dre recombinase

Abstract

HAND2 is a basic helix-loop-helix transcription factor with diverse functions during development. To facilitate the investigation of genetic and functional diversity among Hand2-expressing cells in the mouse, we have generated Hand2^Dre, a knock-in allele expressing Dre recombinase. To avoid disrupting Hand2 function, the Dre cDNA is inserted at the 3' end of the Hand2 coding sequence following a viral 2A peptide. Hand2^Dre homozygotes can therefore be used in complex crosses to increase the proportion of useful genotypes among offspring. Dre expression in mid-gestation Hand2^Dre embryos is indistinguishable from wild-type Hand2 expression, and Hand^Dre efficiently recombines rox target sites in vivo. In combination with existing Cre and Flp mouse lines, Hand2^Dre will therefore extend the ability to perform genetic intersectional labeling, fate mapping, and functional manipulation of subpopulations of cells characterized by developmental expression of Hand2.

Keywords: Dre/rox; heart; limb bud; mouse; neural crest; sympathetic neurons.

Figure 1:. Structure of the Hand2^Dre allele.

A schematic diagram of the Hand2 locus (a) shows the insertion site of the 2A-Dre-poly(A) cassette. Black rectangles represent Hand2 coding sequence. Unfilled rectangles represent 5’ and 3’ UTR extending beyond the limits of the diagram. Partial sequence of wild-type Hand2 exon 2 (b) shows the binding site of the sgRNA on the reverse strand (italicized blue text) and 76 nucleotides deleted in the Hand2^Dre allele (underlined). Upper case text indicates coding sequence, and lower case indicates 3’ UTR. Sequence from the Hand2^Dre allele (c) shows the junction between Hand2, 2A peptide, and Dre cDNA. An exogenous BamHI restriction site (underlined) links Hand2 exon 2 to the 2A peptide. The black arrowhead indicates the cleavage site in the 2A peptide.

Figure 2:. Identical expression patterns of Dre and Hand2 at E10.5 and E14.5.

Labeled riboprobes hybridized to sequential sagittal sections from an E10.5 mouse embryo demonstrate identical Hand2 (a) and Dre (b) expression patterns. Atr, common atrial chamber of heart; BC, bulbus cordis; 1Br, mandibular component of the first branchial arch; hLB, hindlimb bud (see insets); SyC, sympathetic chain. The posterior end of the limb bud, exhibiting higher concentration of Hand2 and Dre mRNA, is uppermost because the embryo was curled. At E14.5 (c-l), spatial expression of the two probes remains identical. To, tongue; Mn, mandible; SCG, superior cervical ganglion; Ht, heart; Vt, wall of ventricle; SyG, sympathetic ganglia; MiG, loops of midgut in the physiological umbilical hernia; LuG, lumen of gut in abdomen; Li, liver; Ki; kidney; Adr, adrenal gland. Scalebar: 1000 μm a-b, 500 μm a-b inset, 2000 μm c-d, 1200 μm e-f, 1000 μm g-h, 680 μm i-j, 685 μm k-l.

Figure 3:. Hand2^Dre recombines a Dre-responsive reporter allele in mouse embryos.

A schematic diagram (a) shows recombination of the RC::RG allele by Hand2^Dre, resulting in expression of enhanced green fluorescent protein (EGFP). Brightfield (b, d) and fluorescence images (c, e) show whole-mount Hand2^Dre; RC::RG double heterozygous embryos at E9.5 and E10.5. EGFP fluorescence indicates tissue with a history of Hand2^Dre expression. Faint fluorescence in the E9.5 head appears to be background autofluorescence because it is absent from the E10.5 tissue. fLB; forelimb bud; hLB, hindlimb bud. Scalebar: 550 μm (b, c); 1000 μm (d, e).