Seamless Ligation Cloning Extract (SLiCE) cloning method.

SLiCE (Seamless Ligation Cloning Extract) is a novel cloning method 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 (15-52 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from bacterial artificial chromosomes or other sources. SLiCE is highly cost-effective and demonstrates the versatility as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. We established a DH10B-derived E. coli strain expressing an optimized λ prophage Red recombination system, termed PPY, which facilitates SLiCE with very high efficiencies.

Germline deletion of Igh 3' regulatory region elements hs 5, 6, 7 (hs5-7) affects B cell-specific regulation, rearrangement, and insulation of the Igh locus.

Regulatory elements located within an ∼28-kb region 3' of the Igh gene cluster (3' regulatory region) are required for class switch recombination and for high levels of IgH expression in plasma cells. We previously defined novel DNase I hypersensitive sites (hs) 5, 6, 7 immediately downstream of this region. The hs 5-7 region (hs5-7) contains a high density of binding sites for CCCTC-binding factor (CTCF), a zinc finger protein associated with mammalian insulator activity, and is an anchor for interactions with CTCF sites flanking the D(H) region. To test the function of hs5-7, we generated mice with an 8-kb deletion encompassing all three hs elements. B cells from hs5-7 knockout (KO) (hs5-7KO) mice showed a modest increase in expression of the nearest downstream gene. In addition, Igh alleles in hs5-7KO mice were in a less contracted configuration compared with wild-type Igh alleles and showed a 2-fold increase in the usage of proximal V(H)7183 gene families. Hs5-7KO mice were essentially indistinguishable from wild-type mice in B cell development, allelic regulation, class switch recombination, and chromosomal looping. We conclude that hs5-7, a high-density CTCF-binding region at the 3' end of the Igh locus, impacts usage of V(H) regions as far as 500 kb away.

SLiCE: a novel bacterial cell extract-based DNA cloning method.

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.

PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance.

The DNA mismatch repair protein PMS2 was recently found to encode a novel endonuclease activity. To determine the biological functions of this activity in mammals, we generated endonuclease-deficient Pms2E702K knock-in mice. Pms2EK/EK mice displayed increased genomic mutation rates and a strong cancer predisposition. In addition, class switch recombination, but not somatic hypermutation, was impaired in Pms2EK/EK B cells, indicating a specific role in Ig diversity. In contrast to Pms2-/- mice, Pms2EK/EK male mice were fertile, indicating that this activity is dispensable in spermatogenesis. Therefore, the PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance and tumor suppression.

Critical function of AP-2 gamma/TCFAP2C in mouse embryonic germ cell maintenance.

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.

Reassessment of the role of Mut S homolog 5 in Ig class switch recombination shows lack of involvement in cis- and trans-switching.

When B cells are activated after immunization or infection, they exchange the gene encoding the Ig H chain C region by class switch recombination (CSR). CSR generally occurs by an intrachromosomal deletional recombination within switch (S) region sequences. However, approximately 10% of CSR events occur between chromosome homologs (trans- or interallele CSR), suggesting that the homologous chromosomes are aligned during CSR. Because the Mut S homolog 4 (Msh4) and Msh5 bind to Holliday junctions and are required for homologous recombination during meiosis in germ cells, we hypothesized these proteins might be involved in trans-chromosomal CSR (trans-CSR). Indeed, Msh4-Msh5 has recently been suggested to have a role in CSR. However, we find a large variety of alternative splice variants of Msh5 mRNA in splenic B cells rather than the full-length form found in testis. Most of these mRNAs are unlikely to be stable, suggesting that Msh5 might not be functional. Furthermore, we find that msh5 nullizygous B cells undergo CSR normally, have unaltered levels of trans-CSR, normal levels of DNA breaks in the Smu region, and normal S-S junctions. We also show that the S-S junctions from cis- and trans-CSR events have similar lengths of junctional microhomology, suggesting trans-CSR occurs by nonhomologous end joining as does intrachromosome (cis)-CSR. From these data, we conclude that Msh5 does not participate in CSR.

Ubiquitylated PCNA plays a role in somatic hypermutation and class-switch recombination and is required for meiotic progression.

Somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes are dependent upon activation-induced cytidine deaminase (AID)-induced mutations. The scaffolding properties of proliferating cell nuclear antigen (PCNA) and ubiquitylation of its residue K164 have been suggested to play an important role organizing the error-prone repair events that contribute to the AID-induced diversification of the Ig locus. We generated knockout mice for PCNA (Pcna(-/-)), which were embryonic lethal. Expression of PCNA with the K164R mutation rescued the lethal phenotype, but the mice (Pcna(-/-)tg(K164R)) displayed a meiotic defect in early pachynema and were sterile. B cells proliferated normally in Pcna(-/-)tg(K164R) mice, but a PCNA-K164R mutation resulted in impaired ex vivo CSR to IgG1 and IgG3, which was associated with reduced mutation frequency at the switch regions and a bias toward blunt junctions. Analysis of the heavy chain V186.2 region after NP-immunization showed in Pcna(-/-)tg(K164R) mice a significant reduction in the mutation frequency of A:T residues in WA motifs preferred by polymerase-eta (Poleta), and a strand-biased increase in the mutation frequency of G residues, preferentially in the context of AID-targeted GYW motifs. The phenotype of Pcna(-/-)tg(K164R) mice supports the idea that ubiquitylation of PCNA participates directly in the meiotic process and the diversification of the Ig locus through class-switch recombination (CSR) and somatic hypermutation (SHM).

Analysis of meiotic prophase I in live mouse spermatocytes.

Events occurring during meiotic prophase I are critical for the successful production of haploid gametes. Many prophase I events are mediated by a meiosis-specific structure called the synaptonemal complex. To date, the limited knowledge we have about the dynamics of these prophase I events in mice comes from fixed, two-dimensional preparations of meiotic cells making it impossible to study the three-dimensional (3D) arrangement of meiotic chromosomes. The current study involves the development of an imaging system to view prophase I events in live mammalian spermatocytes by generating a transgenic mouse, Sycp3-Eyfp ( 21HC ), expressing a fluorescently tagged synaptonemal complex protein, SYCP3. Using this live imaging system, the 3D structural arrangement of chromosomes in the different prophase I substages has been characterized in live spermatocytes, and aspects of the 3D architecture of spermatocytes have been observed that would not be possible with existing techniques. Additionally, chromosome movement in prophase I spermatocytes and meiotic progression from pachynema to diplonema were observed following treatment with the phosphatase inhibitor, okadaic acid (OA), which accelerates the progression of cells through late prophase I. These studies demonstrate that the Sycp3-Eyfp ( 21HC ) live imaging system is a useful tool for the study of mammalian prophase I dynamics.

Inactivation of Tbx1 in the pharyngeal endoderm results in 22q11DS malformations.

The 22q11 deletion (22q11DS; velo-cardio-facial syndrome/DiGeorge syndrome) is characterized by defects in the derivatives of the pharyngeal apparatus. Mouse genetic studies have identified Tbx1, a member of the T-box family of transcription factors, as being responsible for the physical malformations of the syndrome. Mice heterozygous for a null mutation in Tbx1 have mild anomalies, whereas homozygous Tbx1 mutants die at birth with severe defects in the derivatives of the pharyngeal apparatus, including cleft palate, thymus gland aplasia and cardiac outflow tract malformations. Tbx1 is expressed in the splanchnic mesenchyme, the pharyngeal endoderm (PE) and in the core mesoderm of the pharyngeal apparatus. Tissue interactions between the epithelia and mesenchyme of the arches are required for development of the pharyngeal apparatus; the precise role of Tbx1 in each tissue is not known. To assess the role of Tbx1 in the PE, a conditional allele of Tbx1 was generated using the Cre/loxP system. Foxg1-Cre was used to drive PE-specific ablation of Tbx1. Conditional null mutants survived embryogenesis, but died in the neonatal period with malformations identical to the defects observed in Tbx1 homozygous null mutants. The abnormalities appear to be secondary to failed outgrowth of the pharyngeal pouches. These results show that Tbx1 in the PE is required for the patterning and development of the pharyngeal apparatus, thereby disrupting the formation of its derivative structures.

An Msh2 point mutation uncouples DNA mismatch repair and apoptosis.

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.

Transcription factor AP-2gamma stimulates proliferation and apoptosis and impairs differentiation in a transgenic model.

AP-2 transcription factors play pivotal roles in orchestrating embryonic development by influencing the differentiation, proliferation, and survival of cells. Furthermore, AP-2 transcription factors have been implicated in carcinogenesis, a process where the normal growth and differentiation program of cells is disturbed. To experimentally address the potential involvement of AP-2 in mammary gland tumorigenesis, we generated mice overexpressing AP-2gamma by transgenesis using the mouse mammary tumor virus-long terminal repeat as the transgene-driving promoter unit. In the mammary gland, transgene expression elicited a hyperproliferation that, however, was counterbalanced by the enhanced apoptosis of epithelial cells leading to a hypoplasia of the alveolar epithelium during late pregnancy. In addition, secretory differentiation was impaired, resulting in a lactation failure. In male transgenic mice, the seminal vesicles were sites of strong transgene expression. There the effects of AP-2gamma on proliferation and apoptosis were even more pronounced, and differentiation was impaired, too, as revealed by the absence of androgen receptor immunoreactivity. In both tissues, the mammary gland and the seminal vesicles, enhanced steady-state transcript levels of the AP-2 target gene IGFBP-5 were detected, revealing a potential mechanism of AP-2-induced apoptosis. Our results suggest a role of AP-2 transcription factors in the maintenance of a proliferative and undifferentiated state of cells, characteristics not only important during embryonic development but also in tumorigenesis.

Mbd4 inactivation increases Cright-arrowT transition mutations and promotes gastrointestinal tumor formation.

Mbd4 (methyl-CpG binding domain 4) is a novel mammalian repair enzyme that has been implicated biochemically in the repair of mismatched G-T residues at methylated CpG sites. In addition, the human protein has been shown to interact with the DNA mismatch repair protein MLH1. To clarify the role of Mbd4 in DNA repair in vivo and to examine the impact of Mbd4 inactivation on gastrointestinal (GI) tumorigenesis, we introduced a null mutation into the murine Mbd4 gene by gene targeting. Heterozygous and homozygous Mbd4 mutant mice develop normally and do not show increased cancer susceptibility or reduced survival. Although Mbd4 inactivation did not increase microsatellite instability (MSI) in the mouse genome, it did result in a 2- to 3-fold increase in C-->T transition mutations at CpG sequences in splenocytes and epithelial cells of the small intestinal mucosa. The combination of Mbd4 deficiency with a germ line mutation in the adenomatous polyposis coli (Apc) gene increased the tumor number in the GI tract and accelerated tumor progression. The change in the GI cancer phenotype was associated with an increase in somatic C-->T mutations at CpG sites within the coding region of the wild-type Apc allele. These studies indicate that, although inactivation of Mbd4 does not by itself cause cancer predisposition in mice, it can alter the mutation spectrum in cancer cells and modify the cancer predisposition phenotype.

Transcription factor gene AP-2 gamma essential for early murine development.

Transcription factor gene AP-2 gamma belongs to a family of four closely related genes. AP-2 gamma 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 gamma during mammalian development, we generated mice harboring a disrupted AP-2 gamma allele. AP-2 gamma heterozygous mice are viable and display reduced body sizes at birth but are fertile. Mice deficient for AP-2 gamma, 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 gamma 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 gamma 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 gamma seems to be required in early embryonic development because it regulates the genetic programs controlling proliferation and differentiation of extraembryonic trophectodermal cells.