Viral Cre-LoxP tools aid genome engineering in mammalian cells

Background

Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking.

Results

We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions.

Conclusions

We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.

     

Influence of the application of sugarcane bagasse on lindane (gamma-HCH) mobility through soil column: implication for biotreatment

In the present study we employed sugarcane bagasse for biotreatment of soil containing 50mgkg(-1) of lindane. Garden soil were treated with lindane and amended with varying concentrations of sugarcane bagasse (10%, 20%, 30%, 40% and 50%; w/w). Data on dissipation and degradation of lindane in soil columns (0-15, 15-30cm) were studied at six consecutive samplings (0, 3, 7, 45 and 60 days). Treatment with 50% sugarcane bagasse resulted in >53% degradation of lindane in upper soil column with minimal leaching to lower soil column (0.002%) while highest leaching of lindane from upper soil column to lower soil column was occurred in garden soil (35.8%). Similarly, a substantial microbial biomass input has detected in amended soil than garden soil. Our results provide evidence that sugarcane bagasse can accelerate lindane degradation by enhanced microbial activity and prevent pesticide mobility through soil column by adsorption. Sugarcane bagasse could be useful as cheaper, easy available alternative for the biotreatment of lindane impacted soil.     

Metastatic anaplastic oligodendroglioma simulating acute leukemia. A case report

BACKGROUND: Anaplastic oligodendroglioma (OG) is an uncommon tumor that rarely metastasizes outside the central nervous system. Spread to the bone marrow (BM) is so rare that when it occurs in the course of follow-up of a case of OG, a disseminated second primary tumor may be a more likely possibility unless BM examination provides evidence to the contrary. Potentially misleading cytologic features of metastatic anaplastic OG can be seen in a BM touch preparation. CASE: A 50-year-old man had undergone left frontal lobectomy in September 1999 for anaplastic OG and presented seven months later with evidence, on BM scan, of focal abnormal uptake at multiple sites. Bone marrow biopsy confirmed OG secondaries, which, on the touch preparation, appeared not only in clusters but also as single cells, simulating acute leukemia. CONCLUSION: The morphology of anaplastic OG metastatic to BM simulates acute leukemia, as seen on the BM touch preparation. This is relevant particularly in the context of anaplastic OG on follow-up. This diagnostic pitfall can be heightened if a BM aspirate rather than biopsy is performed. Metastatic OG can be added to the list of tumors that metastasize to BM as single cells.     

Purification and characterization of Snf1 kinase complexes containing a defined Beta subunit composition

The Snf1 kinase complex of Saccharomyces cerevisiae contains one of three possible beta subunits encoded by either SIP1, SIP2, or GAL83. Snf1 kinase complexes were purified from cells expressing only one of the three beta subunits using a tandem affinity purification tag on the C terminus of the Snf1 protein. The purified kinase complexes were enzymatically active as judged by their ability to phosphorylate a recombinant protein containing the Snf1-responsive domain of the Mig1 protein. The Snf1 kinase complexes containing Gal83 or Sip2 as the beta subunit showed comparable and high levels of activity, whereas the Sip1-containing enzyme was significantly less active. Examination of the protein composition of the purified Snf1 enzyme complexes indicated that the Sip1 protein was present in substoichiometric levels. Increased gene dosage of SIP1 rescued the ethanol growth defect observed in cells expressing Sip1 as their only beta subunit and increased the in vitro activity of Snf1 kinase purified from these cells. Our studies indicate that the reduced activity of Snf1-Snf4-Sip1 kinase is due to low level of Sip1 accumulation rather than a limited ability of the Sip1 form of the enzyme to direct phosphorylation of specific substrates.     

Linkage analysis of adult height with parent-of-origin effects in the Framingham Heart Study

Current linkage analysis methods for quantitative traits do not usually incorporate imprinting effects. Here, we carried out genome-wide linkage analysis for loci influencing adult height in the Framingham Heart Study subjects using variance components while allowing for imprinting effects. We used a sex-averaged map for the 22 autosomes, while chromosomes 6, 14, 18, and 19 were also analyzed using sex-specific maps. We compared results from these four analyses: 1) non-imprinted with sex-averaged maps, 2) imprinted with sex-averaged maps, 3) non-imprinted with sex-specific maps, and 4) imprinted with sex-specific maps. We found four regions on three chromosomes (14q32, 18p11-q21, 18q21-22, and 19q13) with LOD scores above 2.0, with a maximum LOD score of 3.12, allowing for imprinting and sex-specific maps, at D18S1364 on 18q21. While we obtained significant evidence of imprinting effects in both the 18p11-q21 and 19q13 regions when using sex-averaged maps, there were no significant differences between the imprinted and non-imprinted LOD scores when we used sex-specific maps. Our results illustrate the importance of allowing for gender-specific effects in linkage analyses, whether these are in the form of gender-specific recombination frequencies, or in the form of imprinting effects.     

Streptococcus iniae capsule impairs phagocytic clearance and contributes to virulence in fish

Surface capsular polysaccharides play a critical role in protecting several pathogenic microbes against innate host defenses during infection. Little is known about virulence mechanisms of the fish pathogen Streptococcus iniae, though indirect evidence suggests that capsule could represent an important factor. The putative S. iniae capsule operon contains a homologue of the cpsD gene, which is required for capsule polymerization and export in group B Streptococcus and Streptococcus pneumoniae. To elucidate the role of capsule in the S. iniae infectious process, we deleted cpsD from the genomes of two virulent S. iniae strains by allelic exchange mutagenesis to generate the isogenic capsule-deficient DeltacpsD strains. Compared to wild-type S. iniae, the DeltacpsD mutants had a predicted reduction in buoyancy and cell surface negative charge. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide. Gas-liquid chromatography-mass spectrometry analysis of the S. iniae extracellular polysaccharides showed the presence of l-fucose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, N-acetyl-d-galactosamine, and N-acetyl-d-glucosamine, and all except mannose were reduced in concentration in the isogenic mutant. The DeltacpsD mutants were highly attenuated in vivo in a hybrid striped bass infection challenge despite being more adherent and invasive to fish epithelial cells and more resistant to cationic antimicrobial peptides than wild-type S. iniae. Increased susceptibility of the S. iniae DeltacpsD mutants to phagocytic killing in whole fish blood and by a fish macrophage cell line confirmed the role of capsule in virulence and highlighted its antiphagocytic function. In summary, we report a genetically defined study on the role of capsule in S. iniae virulence and provide preliminary analysis of S. iniae capsular polysaccharide sugar components.     

Mycobacterial PE_PGRS Proteins Contain Calcium-Binding Motifs with Parallel β-roll Folds

The PE_PGRS family of proteins unique to mycobacteria is demonstrated to con- rain multiple calcium-binding and glycine-rich sequence motifs GGXGXD/NXUX. This sequence repeat constitutes a calcium-binding parallel/3-roll or parallel β-helix structure and is found in RTX toxins secreted by many Gram-negative bacteria. It is predicted that the highly homologous PE_PGRS proteins containing multiple copies of the nona-peptide motif could fold into similar calcium-binding structures. The implication of the predicted calcium-binding property of PE_PGRS proteins in the Ught of macrophage-pathogen interaction and pathogenesis is presented.     

Arsenic hazards: strategies for tolerance and remediation by plants

Arsenic toxicity has become a global concern owing to the ever-increasing contamination of water, soil and crops in many regions of the world. To limit the detrimental impact of arsenic compounds, efficient strategies such as phytoremediation are required. Suitable plants include arsenic hyperaccumulating ferns and aquatic plants that are capable of completing their life cycle in the presence of high levels of arsenic through the concerted action of arsenate reduction to arsenite, arsenite complexation, and vacuolar compartmentalization of complexed or inorganic arsenic. Tolerance can also be conferred by lowering arsenic uptake by suppression of phosphate transport activity, a major pathway for arsenate entry. In many unicellular organisms, arsenic tolerance is based on the active removal of cytosolic arsenite while limiting the uptake of arsenate. Recent molecular studies have revealed many of the gene products involved in these processes, providing the tools to improve crop species and to optimize phytoremediation; however, so far only single genes have been manipulated, which has limited progress. We will discuss recent advances and their potential applications, particularly in the context of multigenic engineering approaches.     

Antigenicity and Immunogenicity of a Trimeric Envelope Protein from an Indian Clade C HIV-1 Isolate

Human immunodeficiency virus type 1 (HIV-1) isolates from India mainly belong to clade C and are quite distinct from clade C isolates from Africa in terms of their phylogenetic makeup, serotype, and sensitivity to known human broadly neutralizing monoclonal antibodies. Because many of these properties are associated with the envelope proteins of HIV-1, it is of interest to study the envelope proteins of Indian clade C isolates as part of the ongoing efforts to develop a vaccine against HIV-1. To this end, we purified trimeric uncleaved gp145 of a CCR5 tropic Indian clade C HIV-1 (93IN101) from the conditioned medium of 293 cells. The purified protein was shown to be properly folded with stable structure by circular dichroism. Conformational integrity was further demonstrated by its high affinity binding to soluble CD4, CD4 binding site antibodies such as b12 and VRC01, quaternary epitope-specific antibody PG9, and CD4-induced epitope-specific antibody 17b. Sera from rabbits immunized with gp145 elicited high titer antibodies to various domains of gp120 and neutralized a broad spectrum of clade B and clade C HIV-1 isolates. Similar to other clade B and clade C envelope immunogens, most of the Tier 1 neutralizing activity could be absorbed with the V3-specific peptide. Subsequent boosting of these rabbits with a clade B HIV-1 Bal gp145 resulted in an expanded breadth of neutralization of HIV-1 isolates. The present study strongly supports the inclusion of envelopes from Indian isolates in a future mixture of HIV-1 vaccines.