Gene trap screen to identify mutations in genes expressed in primitive streak and tail bud. pGT1,2,3 vectors are introduced into ES cells, and G418^r colonies, representing integrations in the open reading frames, were selected. The different pGT vectors lack a translation initiation codon but allow selection of integrations in each of the three different reading frames. One set of duplicated colonies is screened for overlapping expression of β-Gal reporter activity (light blue) and T mRNA (purple) after in vitro differentiation, and positive clones are expanded from a master plate. After 5′ RACE PCR to identify endogenous mRNA fused to the vector sequence, ES cell clones corresponding to bona fide integrations in the open reading frame of genes are injected into blastocysts to create chimeric embryos, where expression of β-Gal in the primitive streak is monitored. Lines testing positive are passed through the germ line to create transgenic mice which are used to extensively characterize reporter expression and examine embryonic or postnatal homozygous phenotypes.

Reporter expression in differentiating E148 ES cells and early embryos. (A) Partially overlapping expression of T mRNA (black arrowheads) and β-Gal (white arrowheads) in differentiating E148 ES cells. Approximately 40% of E148 β-Gal-positive cells also express T. β-Gal is localized in the nucleus. (B) Longitudinal section of a 6.5-d.p.c. embryo heterozygous for the E148 integration. β-Gal is strongly expressed in the extraembryonic visceral (black arrowhead) and parietal (white arrowhead) endoderm as well as in the extraembryonic ectoderm (white arrow) and trophoblast (black arrow). Cells in the epiblast (asterisk) express lower levels of β-Gal protein. (C) Posterior view of a whole-mount ∼7.5-d.p.c. embryo, showing high level expression in the trophoblast (arrow) and visceral endoderm (black arrowhead) and lower expression in the primitive streak and node. Scattered cells (white arrowhead) in the embryonic endoderm express high levels of β-Gal protein. Horizontal lines indicate the approximate level and plane of the section shown in panel D. (D) Transverse section of the embryo shown in panel C, showing expression in the visceral endoderm and anterior neurectoderm (asterisk). In the primitive streak (double asterisk), cells in the ectoderm rapidly downregulate reporter expression after ingression to the adjacent mesodermal layer. ps, primitive streak; n, node; m, mesoderm.

Reporter expression in E148 mice during organogenesis and later fetal development. (A) A 9.5-d.p.c. embryo showing strong β-Gal expression in the developing neural tube, optic vesicle, and somites. (B) Enlarged view of the posterior end of a 9.5-d.p.c. embryo, showing expression in the ectodermal layer of the primitive streak. De novo upregulation of β-Gal expression occurs in somites emerging from the presomitic mesoderm. Horizontal lines in panels B and C indicate the limits of somites sII and sIII. A subset of cells throughout the gut, overlapping with the presumed location of primordial germ cells at this stage (16), show intense staining (arrowhead). (C) Enlarged view of the presomitic mesoderm and posterior somites. β-Gal activity is present in the anterior compartment of the nascent somite (s0) and three most recently formed somites (sI to sIII). In more mature somites (sIV and panel A), the level of β-Gal activity is lower and more evenly distributed throughout the somite. (D) Lateral view of a 12.5-d.p.c. embryo showing expression in the cerebral cortex and other neural tissue, in the pinna of the ear, and in somites and limb buds. (E) Enlargement of the embryo shown in panel D. β-Gal expression is high in developing muscle and in cells surrounding the digits. (F) Tail of a 12.5-d.p.c. embryo showing β-Gal activity in somites and the CNH of the tail bud. (G) Detail from a sagittal section of a 15.5-d.p.c. embryo. Expression is strong in the heart and in intercostal muscles, and more moderate expression is found in liver. The box shows the approximate region enlarged in panel K. (H) Sagittal section through the head of a 15.5-d.p.c. embryo, showing β-Gal activity in the ventricular zone, containing progenitors for the cortex, and in a subset of more differentiated cells. The box indicates the approximate region enlarged in panel I. (I) Differentiating cells in the cerebral cortex show cytoplasmic localization of β-Gal protein. (J) In muscle cells, expression is strong and localized primarily to the nucleus. (K) Liver cells show weaker nuclear expression. n, neural tube; s, somite; opv, optic vesicle; psm, presomitic mesoderm; ps, primitive streak; hg, hindgut; c, cerebral cortex; mb, midbrain; drg, dorsal root ganglia; p, pinna; m, muscle; d, digit; a, atrium; v, ventricle of the heart; li, liver; ic, intercostal muscle; vz, ventricular zone.

Cytogenetic mapping of E148 integration. FISH in metaphase chromosome spreads of E148 ES cells with a probe specific to the gene trap vector detects a single chromosomal integration site on the proximal region of chromosome 3 (red). This location was confirmed by using a chromosome 3-specific paint (green).

Jade1 gene structure and multiple-protein alignment of mouse, human, and zebra fish Jade1. (A) Mouse Jade1 is encoded by 10 exons (black boxes 2 to 11). The gene trap insertion occurred between exons 3 and 4. Noncoding exons are shown in numbered grey boxes. Potential polyadenylation signals are indicated by arrows. The box outlined at the end of exon 9 indicates an alternative 3′ untranslated region sequence that would give rise to a ∼3.5-kb transcript and the short Jade1 isoform (Jade1S), equivalent to the published human Jade1 protein (69). EST and cDNA alignments indicate that at least three alternative 5′ noncoding exons (1a to 1c) are used. A promoter scan identified two putative promoter regions in proximity to exons 1a and 1b that coincide with CpG islands. (B) Multiple alignment of mouse Jade1 (Mm) with the putative Jade1 orthologues in human (Hs) and zebra fish (Dr) reveals extensive homology. Residues identical in all three homologues are filled black while those identical only in two of these and conserved amino acid substitutions are filled dark and light grey, respectively. The PHD finger domains are boxed. A potential bipartite NLS is indicated by a dashed underline. Although this NLS is not conserved in the zebra fish, two possible overlapping bipartite NLS are present at positions 635 to 651 and 648 to 664 of DrJade1. Two putative PEST sequences (PESTFIND score > 10) found in both mouse and human Jade1 are underlined in solid black. The position of the gene trap insertion is indicated by the black triangle. The published human Jade1 protein sequence (GenPept accession no. AAM95612) is shorter than the sequence shown here; AAM95612 is identical up to the position marked # below the aligned sequences (corresponding to the end of exon 9) but is truncated at a stop codon (*) after 7 variant amino acids encoded by intron 9.

The Jade protein family. (A) Multiple alignment of mouse Jade1 and the closest related mouse sequences (Jade2 and Jade3) identified in blast searches. Shading is the same as described in the legend to Fig. 5. The PHD fingers are conserved in all three family members (boxed), but there is also extensive conservation outside these domains. (B) A neighbor-joining tree of the Jade protein family. The tree was generated from an alignment of mouse (Mm), human (Hs), zebra fish (Dr), Fugu rubripes (Fr), and Ciona intestinalis (Ci) Jade sequences and rooted with the C. intestinalis sequence. Groups of Jade proteins are indicated by grey rectangles.

Wild-type Jade1 transcripts are absent in embryos homozygous for the E148 integration. Panels A and B show Northern blot analysis of total RNA prepared from 13.5-d.p.c. embryos (lanes 1 to 10) of a heterozygous intercross. The probe detects an mJade1 sequence immediately 3′ to the insertion site. (A) An ethidium bromide-stained gel loading control shows the presence of intact RNA. (B) In wild-type and heterozygous embryos a major ∼6-kb transcript is present (wide arrow). Lower-molecular-size faint bands (indicated by small arrows) are also detected, indicating the presence of smaller minor transcripts, including one at 3.5 kb. No transcripts are detected in homozygous embryos. (C) RT-PCR analysis performed on the same RNA samples with primers from the endogenous Jade1 sequence lying 5′ and 3′ to the E148 insertion (top panel). A wild-type band is detected in all heterozygous and wild-type embryos. A second RT-PCR with the same 5′ primer and a 3′ primer specific to the gene trap vector sequence amplifies a band in all heterozygous and homozygous embryos (bottom panel). No wild-type product is detectable in homozygous mutant embryos (lanes 1, 3, and 9).