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.     

Silencing of the PP2A catalytic subunit causes HER-2/neu positive breast cancer cells to undergo apoptosis

Protein phosphatase 2A (PP2A), in its activated form as a phosphatase, is a tumour suppressor. However, when PP2A is phosphorylated at the tyrosine residue (pY307), it loses its phosphatase activity and becomes inactivated. In our previous study, we found a higher expression of pY307-PP2A in HER-2/neu positive breast tumour samples and significantly correlated to tumour progression, and in this context, it could function as a proto-oncogene. The above and subsequent findings led us to postulate that the critical role of PP2A in maintaining the balance between cell survival and cell death may be linked to its phosphorylation status at its Y307 residue. Hence, we further investigated the effects of knocking down the PP2A catalytic subunit which contains the Y307 amino acid residue in two HER-2/neu positive breast cancer cell lines, BT474 and SKBR3. We showed that this causes the silenced HER-2/neu breast cancer cells to undergo apoptosis and furthermore, that such apoptosis is mediated by p38 MAPK-caspase 3/PARP activation. Understanding the role of PP2A in HER2/neu positive cells might thus provide insight into new targets for breast cancer therapy.     

Comparing system-specific chaperone interactions with their Tat dependent redox enzyme substrates

Redox enzyme substrates of the twin-arginine translocation (Tat) system contain a RR-motif in their leader peptide and require the assistance of chaperones, redox enzyme maturation proteins (REMPs). Here various regions of the RR-containing oxidoreductase subunit (leader peptide, full preprotein with and without a leader cleavage site, mature protein) were assayed for interaction with their REMPs. All REMPs bound their preprotein substrates independent of the cleavage site. Some showed binding to either the leader or mature region, whereas in one case only the preprotein bound its REMP. The absence of Tat also influenced the amount of chaperone-substrate interaction.

Structured summary

MINT-8047497: FdhE (uniprotkb:P13024) and FdoG (uniprotkb:P32176) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046441: HybO (uniprotkb:P69741) and HybE (uniprotkb:P0AAN1) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046375: DmsA (uniprotkb:P18775) and DmsD (uniprotkb:P69853) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046425: TorA (uniprotkb:P33225) and TorD (uniprotkb:P36662) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046393: NarJ (uniprotkb:P0AF26) and NarG (uniprotkb:P09152) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046409: NapD (uniprotkb:P0A9I5) and NapA (uniprotkb:P33937) physically interact (MI:0915) by two hybrid (MI:0018)     

A preliminary study on the function of food begging in Sichuan snub-nosed monkeys (Rhinopithecus roxellana): challenge to begging for nutritional gain

Several hypotheses have been developed to explain what benefits a donor may gain from sharing food with another individual, with nutritional gain assumed to be the sole benefit for the beggar. Recently, it has been proposed that begging behaviour serves a social function in non-human primates. In this study, the nutritional-gain assumption was again challenged based on observations on a captive group of Sichuan snub-nosed monkeys (Rhinopithecus roxellana), or golden snub-nosed monkeys. The major findings from this study are that (1) beggars sometimes left their own branches or passed by available branches to beg for similar food from other individuals, (2) beggars occasionally ignored branches that were acquired by begging and (3) food begging occurred more frequently in the all-male unit after the social rank had changed between 2 individuals in this unit. Overall, these preliminary findings suggest that some begging behaviours in captive golden snub-nosed monkeys were not driven by nutritional gain only; instead, we propose that these begging behaviours could be interpreted as attempts at deriving social benefits.     

Defending the end zone: Studying the players involved in protecting chromosome ends

The linear nature of eukaryotic chromosomes leaves natural DNA ends susceptible to triggering DNA damage responses. Telomeres are specialized nucleoprotein structures that comprise the “end zone” of chromosomes. Besides having specialized sequences and structures, there are six resident proteins at telomeres that play prominent roles in protecting chromosome ends. In this review, we discuss this team of proteins, termed shelterin, and how it is involved in regulating DNA damage signaling, repair and replication at telomeres.     

Dynein mediates retrograde neurofilament transport within axons and anterograde delivery of NFs from perikarya into axons: regulation by multiple phosphorylation events

We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutively-phosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin.     

Genetic dissection of gene regulation in multiple mouse tissues

The analysis of the influence of genetic variation on regulation of gene expression at a near-genome-wide level has become the focus of much recent interest. It is widely appreciated that many genes are expressed in a tissue-specific manner and that others are more ubiquitously expressed but relatively little is known about how genetic variation might influence these tissue patterns of gene expression. In this review we discuss what is known about the tissue specificity of the influence of genetic variation and review the challenges that we face in combining hugely parallel, microarray-based gene analysis with equally expensive genetic analysis. We conclude that the available data suggest that genetic variation is essentially tissue specific in its effects upon gene expression and this has important implications for experimental analysis.     

Epigallocatechin-3-gallate delivers hydrogen peroxide to induce death of ovarian cancer cells and enhances their cisplatin susceptibility

The green tea polyphenol epigallocatechin-3-gallate (EGCG) has cancer chemopreventive properties against various types of cancers. The compound is known to attack various targets in transformed cells. In this report, we examined the action of EGCG on ovarian cancer cells. Eight ovarian cancer cell lines were tested (SKOV3, CAOV3, OVCAR3, OVCAR10, A2780, CP70, C30, and C200) and showed IC50s for EGCG at the micromolar range, including ones that are resistant to the chemotherapeutic drug cisplatin. The ovarian cancer cells were sensitive to H2O2 at similar concentrations, and EGCG treatment led to enhanced intracellular H2O2. Neutralization with pyruvate, a scavenger of H2O2, suggests that the toxicity of EGCG may be mediated by oxidative stress from the free radical. Addition of Tempol, a superoxide dismutase mimetic, demonstrates that H2O2 might be generated endogenously from superoxide. The toxicity of cisplatin and the development of cisplatin resistance are major obstacles in treatment of ovarian cancer. We found that addition of EGCG amplified the toxicity of cisplatin. EGCG increased cisplatin potency by three to six-fold in SKOV3, CAOV3, and C200 cells, the latter being a cell line induced to have several hundred fold resistant to cisplatin above the parental line. Our findings suggest that EGCG may accentuate oxidative stress to inhibit growth of ovarian cancer cells and sensitize them to cisplatin.