Expression of the transgene in targeted cells.

<p>(<b>a</b>) RT-PCR analysis of F9expression in targeted clones. The expected product was amplified from all analyzed homologous recombinants after long-term culture (more than 30 passages), while no transcript was detected in wild-type H9 cells. (<b>b</b>) Western blot of the clone’s lysate using an antibody anti human F9 protein. (<b>c</b>) ELISA analysis of supernatants from recombinant culture. No F9 secretion was detected from wild-type H9 cells. The targeted clones secreted the protein at different levels.</p

Teratoma formation in immunodeficiency mice by targeted cells.

<p>H&E staining of teratomas was performed. Derivatives of all three germ layers were observed in the endoderm: (<b>a</b>) gallbladder, (<b>b</b>) intestinal-like epithelium, and (<b>c</b>) respiratory epithelium; in the mesoderm, (<b>d</b>) cartilage and (<b>e</b>) muscle; and in the ectoderm, (<b>f</b>) squamous epithelium. Bar  = 200 µm.</p

<i>In vitro</i> differentiation of targeted clones.

<p>Immunostaining images show cells derived from all three germ layers, including AFP (endoderm), Tuj1 (ectodermal), and SMA (mesodermal) positive cells. Upon directed differentiation, cell clumps started beating rhythmically, and the expression of Mlc-2a and cTn I revealed that the differentiation into cardiomyocytes in these cells had been completed. Scale bar  = 200 µm (a, c).</p

Gene targeting of the rDNA locus in hESCs.

<p>(<b>a</b>) For hESC transfection, the efficiency was determined by transient nucleofection of H9 single cells by pmaxGFP. (<b>b</b>) Phase-contrast image of a resistant clone just before picking up. (<b>c</b>) After two weeks of drug selection, a portion of resistant clones were picked up and the remaining clones were fixed and stained with Giemsa stain. Clones with diameters of ≥2 millimeters were considered resistant. (<b>d</b>) PCR result using P1 and P2, and DNA sequencing of the PCR product. (<b>e</b>) The targeted clones showed a normal karyotype (46, XX). (<b>f–g</b>) Southern blot analysis showed a 7.1 kb band for targeted clones using a 5′ probe. When using a 3′ probe corresponding to the second exon of <i>F9</i> gene, both a wild type 4.3 kb fragment and an integrated 10.3 kb fragment will be detected. M, marker. Scale bar  = 200 µm (a–b).</p