Transient correction of genetic defects in cultured animal cells by introduction of functional proteins.

Ortiz, Dolores; Baldwin, M M; Lucas, Joseph J.

doi:10.1128/mcb.7.8.3012

Cited by 11 publications

(7 citation statements)

References 15 publications

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“…To examine whether purified Tat retained biological activity, a recently described method, referred to as scrape loading (31,32) (17), demonstrated no increase in LTR-directed CAT activity (Fig. 3B).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Bioassay for trans-activation using purified human immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis.

Gentz

Chen

Rosen

1989

Proc. Natl. Acad. Sci. U.S.A.

113

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Expression of the human immunodeficiency virus tat-encoded protein (Tat) is required for virus replication. A genetic approach was used to facilitate the purification of biologically active Tat. A recombinant Tat protein containing a stretch of six histidine residues and a protease cleavage site was engineered and purified to >95% homogeneity in a single step by immobilized metal-ion chromatography with a special affinity resin that has selectivity for proteins with neighboring histidine residues. A modified scrape loading method for introduction of protein into cell monolayers was used to demonstrate that the purified Tat retained biological activity. Tat function was completely blocked in the presence of transcription inhibitors, which demonstrates the requirement of ongoing mRNA synthesis for trans-activation. These studies indicate that the mechanism of trans-activation is unlikely to involve a direct action of Tat on mRNA stability, transport, or translation and provides the basis for a rapid assay that can be used to identify inhibitors of trans-activation. The methods described herein should be useful for the functional analysis of other proteins that do not confer activity through a receptormediated pathway.Human immunodeficiency virus (HIV) gene expression is tightly controlled by numerous cellular (1, 2) and viral (3-6) trans-acting regulatory factors. The HIV genome alone encodes at least three trans-acting regulatory proteins not present in other animal retroviruses (7-11). It is well established that expression of two of these proteins, referred to as Tat (7) and Rev [regulator of virion expression, previously referred to as art or trs (11)], is required for virus replication (12-15). The tat gene encodes a nucleolar protein (16, 17) that is a positive regulator of virion gene expression. The cisacting control elements responsive to Tat, referred to as TAR (18,19), are present between nucleotides -17 to +44 (18, 19) in the long terminal repeat (LTR) and are thus present in the 5' untranslated leader region of all viral messages. Mechanisms operating at both the transcriptional (18,(20)(21)(22)(23) and posttranscriptional (20,24,25) level have been proposed to explain tat function.The Tat protein contains at least three functional domains (17): (i) a negatively charged region at the amino terminus, (ii) a cysteine-rich region, and (iii) a highly basic region. Studies of tat mutants indicate that the amino-terminal residues and cysteines 22, 25, 27, and 37 are required for Tat function (17). The cysteine-rich region has been proposed to form a metal binding "finger" common to several transcriptional regulatory proteins. Frankel et al. have proposed an alternative structure whereby the cysteines are involved in the formation of a metal-linked dimer (26). The presence of both the highly charged region and the cysteine-rich cluster has hampered protein purification efforts.In the present study we describe a simple and efficient method for purification of biologically active Tat protein.

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Section: Resultsmentioning

confidence: 99%

“…The purified His/Tat protein and the mutant protein containing the Cys-25 mutation were introduced into cell monolayers by using a modified scrape loading procedure (31,32). Briefly, 5 x 105 cells (35-mm dish) were transfected with plasmid pU3R-III (7) and incubated for 40 hr.…”

Section: Methodsmentioning

confidence: 99%

Bioassay for trans-activation using purified human immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis.

Gentz

Chen

Rosen

1989

Proc. Natl. Acad. Sci. U.S.A.

113

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“…Such techniques include lipofection, electroporation, tat-tagging, microinjection, and scrape loading. (9)(10)(11)(12)(13)(14)(15)(16) However, proteins degrade. A more effective technique for treating DC might be the internal generation of the antigen protein in situ within the DC itself.…”

Section: Introductionmentioning

confidence: 99%

Transduction and Utility of the Granulocyte-Macrophage Colony-Stimulating Factor Gene into Monocytes and Dendritic Cells by Adeno-Associated Virus

Liu¹,

Santin²,

Mane³

et al. 2000

Journal of Interferon & Cytokine Research

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The genetic manipulation of antigen-presenting dendritic cells (DC) offers promise for stimulating the immune response, in particular for anticancer and antiviral protocols. As adeno-associated virus (AAV) has shown promise as a gene delivery vector for transducing a variety of hematopoietic cell types, we have investigated AAV's ability to genetically alter DC. In this analysis, we modified the standard granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) treatment of adherent monocytes to generate DC. In our protocol, adherent monocytes were first infected with an AAV/GM-CSF/Neo vector, and the addition of IL-4 was delayed for 2 days to allow for a brief period of monocyte proliferation. AAV-mediated transduction of the GM-CSF and Neo genes into monocytes/DC precursors was demonstrated by G418 selection, GM-CSF secretion, GM-CSF RNA expression (reverse transcriptase-polymerase chain reaction amplification [RT-PCR]), and cell proliferation. Cells resulting from infection with AAV/GM-CSF/Neo virus, and subsequent IL-4 and tumor necrosis factor-alpha (TNF-alpha) treatment, displayed multiple classic markers consistent with mature DC. Finally, chromosomal integration of the AAV vector was also demonstrated in sorted CD83+ DC. These data strongly suggest that AAV vectors will be useful for the genetic manipulation of DC and suggest that the transduction of the GM-CSF gene was able to fully replace the need for exogenous GM-CSF in the production of mature DC.

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“…Electroporation, in contrast, is ideal for bulk delivery applications (3,4); however, general cell damage is also a common consequence (4,5). Sonication, freezing and thawing, osmotic shock, scrape or bead loading, detergent permeabilization, and other forms of cell lysis have also been employed with success in specific applications (6)(7)(8)(9), but the inability to promote adequate resealing in some cells has limited their applicability (10)(11)(12)(13). Erythrocyte-mediated cell fusion has also been used to deliver large amounts of macromolecules directly into the cytoplasm of cultured cells (14,15), but the required use of fusogens, agglutinating lectins, or cytotoxic viruses, as well as the introduction of erythrocyte proteins into the target cells, may yield a system too modified for future study (16)(17)(18).…”

mentioning

confidence: 99%

Delivery of macromolecules into living cells: a method that exploits folate receptor endocytosis.

Leamon

Low

1991

Proc. Natl. Acad. Sci. U.S.A.

533

338

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Difficulties with the nondestructive delivery of macromolecules into living cells have limited the potential applications of antibodies, genes, enzymes, peptides, and antisense oligonucleotides in biology and medicine. We have found, however, that the natural endocytosis pathway for the vitamin folate can be exploited to nondestructively introduce macromolecules into cultured cells if the macromolecule is first covalently linked to folate. Thus, treatment of KB cells with folate-conjugated ribonuclease, horseradish peroxidase, serum albumin, IgG, or ferritin allowed delivery of >106 copies of the macromolecules within a 2-hr period. Cytochemical staining using 4-chloro-1-naphthol further demonstrated that the horseradish peroxidase retained activity for at least 6 hr after internalization. Since folate is an essential vitamin required in substantial quantities by virtually all cells, these observations may open the possibility of scientific and medical applications for many of the above macromolecules.

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Transient correction of genetic defects in cultured animal cells by introduction of functional proteins.

Cited by 11 publications

References 15 publications

Bioassay for trans-activation using purified human immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis.

Bioassay for trans-activation using purified human immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis.

Transduction and Utility of the Granulocyte-Macrophage Colony-Stimulating Factor Gene into Monocytes and Dendritic Cells by Adeno-Associated Virus

Delivery of macromolecules into living cells: a method that exploits folate receptor endocytosis.

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