Generation of a triple‐gene knockout mammalian cell line using engineered zinc‐finger nucleases

Mammalian cells with multi‐gene knockouts could be of considerable utility in research, drug discovery, and cell‐based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events—a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc‐finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi‐locus genome engineering. Biotechnol. Bioeng. 2010; 106: 97–105. © 2009 Wiley Periodicals, Inc.     

Role of the p63-FoxN1 regulatory axis in thymic epithelial cell homeostasis during aging

The p63 gene regulates thymic epithelial cell (TEC) proliferation, whereas FoxN1 regulates their differentiation. However, their collaborative role in the regulation of TEC homeostasis during thymic aging is largely unknown. In murine models, the proportion of TAp63⁺, but not ΔNp63⁺, TECs was increased with age, which was associated with an age-related increase in senescent cell clusters, characterized by SA-β-Gal⁺ and p21⁺ cells. Intrathymic infusion of exogenous TAp63 cDNA into young wild-type (WT) mice led to an increase in senescent cell clusters. Blockade of TEC differentiation via conditional FoxN1 gene knockout accelerated the appearance of this phenotype to early middle age, whereas intrathymic infusion of exogenous FoxN1 cDNA into aged WT mice brought only a modest reduction in the proportion of TAp63⁺ TECs, but an increase in ΔNp63⁺ TECs in the partially rejuvenated thymus. Meanwhile, we found that the increased TAp63⁺ population contained a high proportion of phosphorylated-p53 TECs, which may be involved in the induction of cellular senescence. Thus, TAp63 levels are positively correlated with TEC senescence but inversely correlated with expression of FoxN1 and FoxN1-regulated TEC differentiation. Thereby, the p63-FoxN1 regulatory axis in regulation of postnatal TEC homeostasis has been revealed.     

Placenta‐specific gene activation and inactivation using integrase‐defective lentiviral vectors with the Cre/LoxP system

Transgenic and knockout studies have advanced our understanding of the genetic control of embryonic development over the past decades. However, interpretation of the phenotype of mutant mice is potentially complicated, since the commonly used knockout approach modifies both the fetal and placental genome. To circumvent this problem, we previously developed a placenta‐specific gene manipulation system by lentiviral vector transduction of embryos at the blastocyst stage. In the present study, by combination with the Cre/LoxP system, we successfully demonstrate placenta‐specific gene activation and inactivation in EGFP reporter mice and Ets2 floxed mice, respectively. Transient expression using integrase‐defective lentiviral (IDLV) vectors diminished the toxic effect of Cre expression and solved the dilemma of mosaic recombination with lower concentrations and toxic effects with higher concentrations of Cre recombinase. We also show that placenta‐specific Ets2 disruption causes embryonic lethality and reconfirmed the critical role of Ets2 during placentation. This technology facilitates both gain and loss of gene function analyses in placental development during pregnancy. Since IDLV vectors can efficiently transduce a variety of cell types similarly to wild‐type vectors, our IDLV‐Cre strategy is potentially useful for a wide range of applications. genesis 47:793–798, 2009. © 2009 Wiley‐Liss, Inc.     

Induction of the IFN-gamma gene and protein is linked to the establishment of cell polarity in a porcine epithelial cell line

We report here original properties of a porcine trophectoderm cell line, TBA B4-3, that developed a polarized phenotype with high transepithelial electrical resistance (TER) values and functional tight junctions (TJs) when grown on a microporous membrane. We found that treatment of polarized TBA B4-3 cells with a strong protein kinase C (PKC) agonist, phorbol 12-myristate-13-acetate (PMA), induced 3-4 days later a transient interferon-gamma (IFN-gamma) mRNA expression and vectorial IFN-gamma protein secretion toward the apical side of the monolayer. Exposure of TBA B4-3 cells to PMA first resulted in a rapid and profound disorganization of the monolayer structure mainly characterized by the appearance of multilayered polyp-like foci structures, a strong decrease of the TER, and a increase of permeability correlated with changes in the organization and localization of the TJ-associated proteins (ZO-1 and occludin) and filamentous actin (f-actin). After PMA removal, spontaneous return to the initial polarized monolayer state occurred, characterized by TER rising to prestimulation values, TJ protein relocalization, and multilayered cell structures fading. This return was strictly correlated with transient IFN-gamma gene induction. Our report represents the first example of an inducible expression of IFN-gamma by a polarized epithelial cell. After PMA treatment, the close correlation between establishment of cell polarity and IFN-gamma gene expression suggests a link between these phenomena. This also suggests a novel biological mechanism by which transient and reversible disorganization of a polarized monolayer of epithelial cells could trigger regulated expression of a cytokine gene by these cells.     

Disruption of the mouse protein Ser/Thr phosphatase 2Cbeta gene leads to early pre-implantation lethality

Protein phosphatase 2Cβ (PP2Cβ) is a member of a family of protein Ser/Thr phosphatases (PP2C) that is composed of at least twelve different gene products. Recent studies have revealed that PP2Cβ mRNA accumulates in mature sperm, unfertilized metaphase II-arrested oocytes and zygotes, but that the mRNA level then decreases sharply between the early two-cell and eight-cell stages, remaining at low levels during the 16-cell to blastocyst stages of mice. These observations raised the possibility that PP2Cβ plays a crucial role during gametogenesis, fertilization, and/or early stages of embryonic development. In this study, we employed a gene knockout technique in mice to test this possibility. We found that PP2Cβ^Δ/wt mice generate normal mature gametes. However, PP2Cβ^Δ/Δ embryos die between the two-cell and eight-cell stages. To our interest, PP2Cβ^Δ/Δ ES cells which had been generated by transfecting PP2Cβ^3lox/3lox ES cells with Cre-expressing plasmid were viable. In addition, knockdown of PP2Cβ using siRNA did not affect the proliferation of wild-type ES cells. These observations suggest that relatively high PP2Cβ expression is specifically required during the early stages of pre-implantation development. The possible mechanisms for the early pre-implantation lethality of PP2Cβ^Δ/Δ mice are discussed.     

Novel crosstalk between ERK MAPK and p38 MAPK leads to homocysteine‐NMDA receptor‐mediated neuronal cell death

Hyperhomocysteinemia is an independent risk factor for both acute and chronic neurological disorders, but little is known about the underlying mechanisms by which elevated homocysteine can promote neuronal cell death. We recently established a role for NMDA receptor‐mediated activation of extracellular signal‐regulated kinase (ERK)‐MAPK in homocysteine‐induced neuronal cell death. In this study, we examined the involvement of the stress‐induced MAPK, p38 in homocysteine‐induced neuronal cell death, and further explored the relationship between the two MAPKs, ERK and p38, in triggering cell death. Homocysteine‐mediated NMDA receptor stimulation and subsequent Ca²⁺ influx led to a biphasic activation of p38 MAPK characterized by an initial rapid, but transient activation followed by a delayed and more prolonged response. Selective inhibition of the delayed p38 MAPK activity was sufficient to attenuate homocysteine‐induced neuronal cell death. Using pharmacological and RNAi approaches, we further demonstrated that both the initial and delayed activation of p38 MAPK is downstream of, and dependent on activation of ERK MAPK. Our findings highlight a novel interplay between ERK and p38 MAPK in homocysteine‐NMDA receptor‐induced neuronal cell death.     

Resistance of mitochondrial DNA to degradation characterizes the apoptotic but not the necrotic mode of human leukemia cell death

Cell death can occur by two basically different processes. The original term, necrosis, is now reserved for the generally destructive series of events which include the release of lysosomal enzymes and loss of cell membrane integrity. In contrast, mild treatment with cell damaging agents, or withdrawal of growth factors, may result in a characteristic form of degradation of cellular DNA which is associated with cell death that has morphology known as apoptosis. In this study human leukemia cells were exposed to agents or conditions previously reported to cause necrosis or apoptosis, monitored by detection of DNA “ladders,” and the integrity of cellular DNA was determined on Southern blots. Nuclear DNA was distinguished from mitochondrial DNA by use of probes specific for nuclear genes or for mitochondrial DNA. When HL60, K562, MOLT4, or U937 cells were exposed to conditions which resulted in necrosis, mitochondrial DNA was damaged at approximately the same rate as nuclear DNA, but in apoptosis mtDNA was not degraded. Thus, the ratio of the relative (to untreated cells) abundance of mitochondrial DNA measured by a probe for 16S mitochondrial ribosomal RNA on Southern blots, to the relative abundance of DNA of any nuclear gene, was 1 or less in necrosis, but rose to values greater than 2 in apoptosis. It is concluded that the comparison of the degree of fragmentation of mitochondrial and nuclear DNA provides a quantitative way of distinguishing necrosis from apoptosis.     

Suppression of the alpha-isoform of class II phosphoinositide 3-kinase gene expression leads to apoptotic cell death

Phosphoinositide 3-kinases (PI3Ks) have known to be key enzymes activating intracellular signaling molecules when a number of growth factors bind to their cell surface receptors. PI3Ks are divided into three classes (I, II, and III) and enzymes of each class have different tissue-specificities and physiological functions. Class II PI3Ks consist of three isoforms (alpha,beta,gamma). Although the alpha-isoform (PI3K-C2alpha) is considered ubiquitous and preferentially activated by insulin rather than the beta-isoform, the physiological significance of PI3K-C2alpha is poorly understood. The present study aimed to determine whether PI3K-C2alpha is associated with the suppression of apoptotic cell death. Different sense- and antisense oligonucleotides (ODNs) were synthesized based on the sequence of C2 domain of PI3K-C2alpha gene. Transfection of CHO-IR cells with two different antisense ODNs clearly reduced the protein content as well as mRNA levels of PI3K-C2alpha whereas neither the nonspecific mock- nor sense ODNs affected. The decrease of PI3K-C2alpha gene expression was paralleled by cellular changes indicating apoptotic cell death such as nuclear condensation, formation of apoptotic bodies, and DNA fragmentation. PI3K-C2alpha mRNA levels were also reduced when cells were incubated in growth factor-deficient medium. Supplementing growth factors (serum or insulin) into medium lead to an increase of PI3K-C2alpha mRNA levels. This finding strongly suggests that PI3K-C2alpha is a crucial survival factor.