We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.

Mutations in the human DNA mismatch repair gene MSH2 are associated with hereditary nonpolyposis colorectal cancer as well as a significant proportion of sporadic colorectal cancer. The inactivation of MSH2 results in the accumulation of somatic mutations in the genome of tumor cells and resistance to the genotoxic effects of a variety of chemotherapeutic agents. Here we show that the DNA repair and DNA damage-induced apoptosis functions of Msh2 can be uncoupled using mice that carry the G674A missense mutation in the conserved ATPase domain. As a consequence, although Msh2 G674A homozygous mutant mice are highly tumor prone, the onset of tumorigenesis is delayed as compared with Msh2-null mice. In addition, tumors that carry the mutant allele remain responsive to treatment with a chemotherapeutic agent. Our results indicate that Msh2-mediated apoptosis is an important component of tumor suppression and that certain MSH2 missense mutations can cause mismatch repair deficiency while retaining the signaling functions that confer sensitivity to chemotherapeutic agents.

Formation of the germ cell lineage involves multiple processes, including repression of somatic differentiation and reacquisition of pluripotency as well as a unique epigenetic constitution. The transcriptional regulator Prdm1 has been identified as a main coordinator of this process, controlling epigenetic modification and gene expression. Here we report on the expression pattern of the transcription factor Tcfap2c, a putative downstream target of Prdm1, during normal mouse embryogenesis and the consequences of its specific loss in primordial germ cells (PGCs) and their derivatives. Tcfap2c is expressed in PGCs from Embryonic Day 7.25 (E 7.25) up to E 12.5, and targeted disruption resulted in sterile animals, both male and female. In the mutant animals, PGCs were specified but were lost around E 8.0. PGCs generated in vitro from embryonic stem cells lacking TCFAP2C displayed induction of Prdm1 and Dppa3. Upregulation of Hoxa1, Hoxb1, and T together with lack of expression of germ cell markers such Nanos3, Dazl, and Mutyh suggested that the somatic gene program is induced in TCFAP2C-deficient PGCs. Repression of TCFAP2C in TCam-2, a human PGC-resembling seminoma cell line, resulted in specific upregulation of HOXA1, HOXB1, MYOD1, and HAND1, indicative of mesodermal differentiation. Expression of genes indicative of ectodermal, endodermal, or extraembryonic differentiation, as well as the finding of no change to epigenetic modifications, suggested control by other factors. Our results implicate Tcfap2c as an important effector of Prdm1 activity that is required for PGC maintenance, most likely mediating Prdm1-induced suppression of mesodermal differentiation.

Transcription factor gene AP-2␥ belongs to a family of four closely related genes. AP-2␥ had been implicated in multiple functions during proliferation and differentiation based on its expression pattern in trophoblast, neural crest, and ectoderm cells in murine embryos. In order to address the question of the role of AP-2␥ during mammalian development, we generated mice harboring a disrupted AP-2␥ allele. AP-2␥ heterozygous mice are viable and display reduced body sizes at birth but are fertile. Mice deficient for AP-2␥, however, are growth retarded and die at days 7 to 9 of embryonic development. Immunohistochemical analysis revealed that the trophectodermal cells that are found to express AP-2␥ fail to proliferate, leading to failure of labyrinth layer formation. As a consequence, the developing embryo suffers from malnutrition and dies. Analysis of embryo cultures suggests that AP-2␥ is also implicated in the regulation of the adenosine deaminase (ADA) gene, a gene involved in purine metabolism found expressed at the maternal-fetal interface. Therefore, AP-2␥ seems to be required in early embryonic development because it regulates the genetic programs controlling proliferation and differentiation of extraembryonic trophectodermal cells.The AP-2 transcription factor gene family consists of four different genes referred to as AP-2␣, AP-2␤, AP-2␥, and the recently discovered AP-2␦ gene (4,15,17,19,22,37,40). All members of the AP-2 family share a characteristic protein structure containing a unique C-terminal helix-span-helix motif that mediates protein dimerization; together with a basic domain they are involved in DNA binding with varying affinity to GC-rich elements. An N-terminal proline-and glutaminerich region mediates transcriptional activation (33,36).Despite the expression of all AP-2 genes in the extraembryonic trophoblast cells, it remained unclear if there was a redundant function in placental gene regulation. Gene knockout experiments with AP-2␣ and AP-2␤ indicate that the AP-2 proteins carry out individual functions during mouse development. While AP-2␣ is predominantly essential for craniofacial development and ventral body wall closure (29, 39), lack of AP-2␤ leads to polycystic kidney disease (20). Both AP-2␣-and AP-2␤-deficient mice display unaltered implantation and placentation. Is AP-2␥ a key regulator of placental development? Prior to implantation, AP-2␥ is expressed in the trophoblast cells starting at day 3.5 of murine development. After implantation, the expression of AP-2␥ continues in the trophoblast cells and its derivatives, the primary giant cells and the diploid cells of the polar trophectoderm. With ongoing proliferation of the trophoblast cells, AP-2␥ is expressed in the ectoplacental cone and the extraembryonic ectoderm. At the time of chorioallantoic fusion, AP-2␥ expression is increased in all derivatives of the trophoblast lineage (27, 30). Thus, AP-2␥, together with the T-box gene Eomes (26), is the only transcription factor gene found to be expressed in all trophoblast li...

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