A nuclear transformation system has been developed for the diatomPhaeodactylum tricornutum using microparticle bombardment to introduce thesh ble gene fromStreptoalloteichus hindustanus into cells. Thesh ble gene encodes a protein that confers resistance to the antibiotics Zeocin and phleomycin. Chimeric genes containing promoter and terminator sequences from theP. tricornutum fcp genes were used to drive expression ofsh ble. Between 10–100 transformants were recovered/108 cells. Transformants were able to grow on at least 500 µg/ml of Zeocin, which is 10 times the amount necessary to kill wild-type cells. Based on Southern hybridizations thesh ble gene was present in 1–3 copies/transformant. Relative levels of correctly processed transcripts were correlated with the abundance of the Sh ble protein (present at 0.1–2.0 µg/mg total protein). Thecat reporter gene fused to afcp promoter could also be introduced by microparticle bombardment and was found to be highly expressed (average of 7.1 U/mg total protein). This work demonstrates that heterologous genes can be readily expressed inP. tricornutum. The development of selectable marker and reporter gene constructs provides the tools necessary for dissecting gene structure and regulation, and introducing novel functions into diatoms.

Stable nuclear transformation of the diatom Phaeodactylum tricornutum.

Transformation methods are provided for introducing deoxyribonucleic acid (DNA) into the nucleus of an algal cell. A transformation construct may be prepared, with the transformation construct having a first sequence of DNA similar to a corresponding first sequence of nuclear DNA, a second sequence of DNA similar to a corresponding second sequence of the nuclear DNA, and a sequence of DNA inserted between the first and second sequences of DNA of the transformation construct. A target sequence of DNA inserted between the first and second corresponding sequences of the nuclear DNA may be transformed, resulting result in replacement of the target sequence of DNA with the sequence of DNA of interest.

https://www.pnas.org/content/pnas/108/52/21265.full.pdf

High-efficiency homologous recombination in the oil-producing alga, nannochloropsis

A general purpose transformation vector, designated pPha-T1, was constructed for use with the diatom Phaeodactylum tricornutum Bohlin. This vector harbors the sh ble cassette for primary selection on medium containing the antibiotic zeocin, and a multiple cloning site flanked by the P. tricornutum fcpA promoter. pPha-T1 was used to establish the utility of three selectable marker genes and two reporter genes for P. tricornutum transformation. The nat and sat-1 genes confer resistance to the antibiotic nourseothricin, and nptII confers resistance to G418. Each of these genes was effective as a selectable marker for identifying primary transformants. These markers could also be used for dual selections in combination with the sh ble gene. The reporter genes uidA and gfp were also introduced into P. tricornutum using pPha-T1. Gus expression in some transformants reached 15 μg·μg−1 of total soluble protein and permitted excellent cell staining, while GFP fluorescence was readily visible with standard fluorescence microscopy. The egfp gene, which has optimal codon usage for expression in human cells, was the only version of gfp that produced a strong fluorescent signal in P. tricornutum. The codon bias of the egfp gene is similar to that of P. tricornutum genes. This study suggests that codon usage has a significant effect on the efficient expression of reporter genes in P. tricornutum. The results presented here demonstrate that a variety of selectable markers and reporter genes can be expressed in P. tricornutum, enhancing the potential of this organism for exploring basic biological questions and industrial applications.

Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes

Development of defective and persistent Sendai virus vector: a unique gene delivery/expression system ideal for cell reprogramming.

Vectors based on oncoretroviruses such as Moloney murine leukemia virus (MoMLV) are useful to deliver therapeutic genes into primary cells in vitro and have also been applied in a number of gene marking and gene therapy trials with humans (13, 62). The principal advantages of retroviral vectors include the high efficiency of gene delivery, integration into the host cell genome, and high level of gene expression. One drawback of oncoretroviruses is their dependence on cell proliferation for completion of the life cycle (35, 56). Breakdown of the nuclear envelope that accompanies mitosis appears to be essential for the import of the viral preintegration complex into the nucleus and its integration into the genome of the host cell (26, 27, 51). In contrast, lentiviruses, including human immunodeficiency virus type 1 (HIV-1), differ fundamentally from oncoretroviruses in that they are independent of cell division for completion of their replicative cycle (58). This is an attractive feature in view of the need for vectors for nondividing cells such as neurons (14, 25). In common with all replication-competent retroviruses, the HIV-1 genome contains the gag, pol, and env coding regions, which encode the core proteins, the virion-associated enzymes, and the envelope (Env) glycoprotein, respectively, flanked by the long terminal repeats (LTRs). The LTRs include cis-acting sequences required for integration, transcription, and polyadenylation. HIV-1 also possesses regulatory functions encoded by the tat and rev genes as well as accessory genes that include vif, vpr, vpu, and nef, many of which are not required for virus replication in vitro (17).

Another advantage of HIV-1 and other retroviruses is the fact that they can be pseudotyped by the incorporation of heterologous glycoproteins, allowing an extension of the host range of such vectors beyond cells expressing CD4. Several studies have demonstrated that HIV-1 produced in cells infected with xenotropic murine leukemia virus (6, 30), amphotropic murine leukemia virus (9, 55), or herpes simplex virus (64) gave rise to phenotypically mixed virions with an expanded host range, suggesting that pseudotyped virions had formed. Additionally, phenotypic mixing of viral envelopes was shown to occur between HIV-1 and vesicular stomatitis virus (VSV) in coinfected cell cultures (64). Page et al. (40) showed that expression of amphotropic or ecotropic MoMLV Env glycoproteins in cells transfected with an HIV-1 vector construct produced virus capable of infecting both human and murine cells, and Landau et al. (23) demonstrated that HIV-1 efficiently incorporated the human T-cell leukemia virus type 1 Env. These observations were confirmed and extended by results showing that the VSV G glycoprotein (VSV-G) was efficiently incorporated into HIV-1 virions, with pseudotyped viral titers reaching 107 CFU/ml or higher (2, 38, 49).

A number of replication-defective HIV-1 vector systems have been described. With the original transient two-plasmid expression system (23, 40), titers of up to 2 × 105 CFU/ml were obtained on human osteosarcoma (HOS) cells. Several groups have designed transient three-component HIV-1 expression systems consisting of a packaging construct, a plasmid bearing the gene encoding gp160, and an expression vector carrying a reporter gene (5, 41, 42, 45, 50, 53, 59), thus reducing the likelihood of generating replication-competent virus. In general, these systems were quite inefficient, with titers of around 104 CFU/ml or below. A number of studies have dealt with the design of HIV-1-based packaging cell lines. While the initial vector titers were quite low (7), recent improvements which involve tetracycline-controlled HIV-1-based packaging constructs (63), different molecular clones (11, 44), or Rev-independent cell lines (57) suggest that the generation of high-titer HIV-1-based packaging cell lines will eventually be feasible. Improved three-component split packaging systems were recently described by Naldini et al. (38) and Kim et al. (21). In these systems, the viral particles were pseudotyped with the envelope of VSV. Titers of up to 9 × 105 transducing units per ml were obtained. In this report, we describe an improved and versatile high-titer three-plasmid-based packaging system and demonstrate its applicability for the efficient transduction of nondividing cells, including growth-arrested HOS cells, confluent primary human skin fibroblasts (HSFs), and postmitotic rat cardiac myocytes and cerebellar neurons.

https://jvi.asm.org/content/jvi/72/11/8873.full.pdf

High-Titer Human Immunodeficiency Virus Type 1-Based Vector Systems for Gene Delivery into Nondividing Cells

Phleomycins are glycopeptide antibiotics related to bleomycins nucleotide sequence of the Sh ble gene and its polylinker and tallysomycins which are active at low concentrations on both fragments. prokaryotic and eukaryotic cells (1). We have cloned a phleomycin-resistance gene (Sh ble) from the genomic DNA of REFERENCES Streptoalloteichus hindustanus (ATCC 31158), a tallysomycin 1 Berdy,J. (1980) Handbook of.Antibiotic Compounds. Vol.4, pp. 459-491, producer. The Sh ble gene encodes a small acidic protein (MW CRC Press, Boca Raton, FL, USA 13665) which has been crystallised for X-ray structural studies 2. Rondeau,J.M., Cagnon,C., Moras,D. and Masson,J.M. (1989) J. MoI. Biol. (2). The Sh protein is a binding protein with a strong affinity 207, 645-646. for the phleomycin family of antibiotics (3). When these 3. Gatignol,A., Durand,H. and Tiraby,G. (1988) FEBS Lent. 230, 171-175. antibiotics are bound by the Sh protein, then phleomycins can 4. Glumoff,V., Kappeli,O., Fiechter,A. and Reiser,J. (1989) Gene 84, nolonger areactivated by tneffous ions and oxyen teobreakdcan 311-318. no longer be activated by ferrous ions and oxygen to break down 5. Mattern,J.E. and Punt,P.J. (1988) Fungal Genet. Newslett. 35, 25. DNA (3). The Sh ble gene has been used in a number of 6. Perez,P., Tiraby,G., Kallerhoff,J. and Perret,J. (1989) Plant Mol. Biol. 13, laboratories as a selectable marker for lower (4, 5) and higher 365-373. eukaryotes (6, 7, 8). In order to facilitate vector construction 7. Mulsant,P., Gatignol,A., Dalens,M. and Tiraby,G. (1988) Somat. Cell. Mol. exeukryoetes(, 8).theinordgoers wto fac tipltevctoricositruction Genet. 14, 243-252. periment , synthetic oligom with multiple cloning sites have 8. Cosset,F.L., Legras,C., Chebloune,Y., Savater,P., Thoraval,P., been added at both ends of the Sh ble gene to give the two pUC Thomas,J.L., Samarut,J., Nigon,U.M. and Verdier,G. (1990) J. Virol. 64, based derivatives, pUT56 and pUT58. We report here the 1070-1078.

Cassettes of the Streptoalloteichus hindustanus ble gene for transformation of lower and higher eukaryotes to phleomycin resistance

The prodrug activation system formed by the E. coli codA gene encoding cytosine deaminase (CD) and 5-fluorocytosine (5-FC) developed for selective cancer chemotherapy suffers from a sensitivity limitation in many tumour cells. In an attempt to improve the CD/5-FC suicide association, we combined the E. coli upp gene encoding uracil phosphoribosyltransferase (UPRT) with codA gene to create the situation prevailing in E. coli, a bacterium very efficient in metabolising 5-FC. The constitutive expression of the two genes cloned on an E. coli-animal cell shuttle plasmid either in a linked or in a fused configuration was evaluated in E. coli strains selected and engineered to mimic the 5-FC metabolism encountered in mammalian cells. The simultaneous expression of codA and upp genes generated a cooperative effect resulting in a dramatic increase in 5-FC sensitivity of cells compared to the expression of codA alone. Furthermore, it was shown that the association of UPRT with CD facilitated the uptake of 5-FC, in the situation where the drug penetrates cells by passive diffusion as in mammalian cells, by directly channeling 5-fluorouracil, the product of CD, to 5-fluoroUMP, the product of UPRT.

Concomitant expression of E. coli cytosine deaminase and uracil phosphoribosyltransferase improves the cytotoxicity of 5‐fluorocytosine

A selectable bifunctional β-galactosidase::phleomycin-resistance fusion protein as a potential marker for eukaryotic cells

Two groups of hybrid suicide genes are disclosed, where the genes of one group specifically activate 5-fluorocytosine, a pyrimidine nucleobase analogue, while the genes of the other group activate azidothymidine, a pyrimidine nucleobase analogue, into derivatives toxic for mammalian cells. A variety of methods are also disclosed for selectively killing transfected tumour cells or immune cells using a single hybrid suicide gene or a combination of two hybrid suicide genes selected for the complementarity of their antimetabolic activities. Furthermore, eukaryotic vectors including two suicide gene expression units, i.e. a first unit sensitising the tumour cells to 5-fluorocytosine or 5-fluorouracil, and a second making HIV-infected cells synergistically resistant to azidothymidine.

Suicide genes and combinations of pyrimidine nucleoside and nucleobase analogues with suicide genes for gene therapy

Gene transfer into cells or tissue by application of electric pulses (i.e. gene electrotransfer (GET)) is a non-viral gene delivery method that is becoming increasingly attractive for clinical applications. In order to make GET progress to wide clinical usage its efficacy needs to be improved and the safety of the method has to be confirmed. Therefore, the aim of our study was to increase GET efficacy in skin, by optimizing electric pulse parameters and the design of electrodes. We evaluated the safety of our novel approach by assaying the thermal stress effect of GET conditions and the biodistribution of a cytokine expressing plasmid. Transfection efficacy of different pulse parameters was determined using two reporter genes encoding for the green fluorescent protein (GFP) and the tdTomato fluorescent protein, respectively. GET was performed using non-invasive contact electrodes immediately after intradermal injection of plasmid DNA into mouse skin. Fluorescence imaging of transfected skin showed that a sophistication in the pulse parameters could be selected to get greater transfection efficacy in comparison to the standard ones. Delivery of electric pulses only mildly induced expression of the heat shock protein Hsp70 in a luminescent reporting transgenic mouse model, demonstrating that there were no drastic stress effects. The plasmid was not detected in other organs and was found only at the site of treatment for a limited period of time. In conclusion, we set up a novel approach for GET combining new electric field parameters with high voltage short pulses and medium voltage long pulses using contact electrodes, to obtain a high expression of both fluorescent reporter and therapeutic genes while showing full safety in living animals.

/pdf/safe-and-efficient-novel-approach-for-non-invasive-gene-4l197nssoh.pdf

Jean-Paul Reynes

Papers

Cassettes of the Streptoalloteichus hindustanus ble gene for transformation of lower and higher eukaryotes to phleomycin resistance

Concomitant expression of E. coli cytosine deaminase and uracil phosphoribosyltransferase improves the cytotoxicity of 5‐fluorocytosine

A selectable bifunctional β-galactosidase::phleomycin-resistance fusion protein as a potential marker for eukaryotic cells

Suicide genes and combinations of pyrimidine nucleoside and nucleobase analogues with suicide genes for gene therapy

Safe and efficient novel approach for non-invasive gene electrotransfer to skin.