Binding of large liposomes to plant protoplasts and delivery of encapsulated DNA

Lurquin, Paul F.; Sheehy, Raymond E.

doi:10.1016/0304-4211(82)90171-7

Cited by 29 publications

(10 citation statements)

References 20 publications

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“…However, the low frequency of transformation observed precludes this system from being used in the evaluation of the effectiveness of DNA transfer. Therefore, an estimation of donor DNA transfer to recipient nuclei presently has to rely on autoradiographic or biochemical techniques (9,12,15,20), even though the results of such experiments are not absolutely quantitative (10). Our results showed that nuclei isolated from protoplasts incubated with DNA-loaded targeted liposomes contained much more donor DNA than if control unmodified liposomes were used.…”

Section: Discussionmentioning

confidence: 82%

“…Protoplasts were produced by incubating cells in high salt medium (2.5% KCI, 0.15% CaCl2, 0.1% MgCli) containing 1% Cellulysin (Calbiochem) and 0.5% Macerase (Calbiochem) for 4 h at 29°C. In some experiments, carrot (Daucus carota L.) cells grown in suspension culture (20) were used to produce protoplasts as described before (12,20) except that high salt medium was used instead of mannitol. All protoplasts were washed three times with high salt medium prior to use.…”

Section: Methodsmentioning

confidence: 99%

“…Positively charged multilamellar liposomes were generated by mechanical shaking as extensively described before (9,12). They were composed of L-a-phosphatidylcholine and stearylamine in a 10:1 (w/w) ratio.…”

Section: Methodsmentioning

confidence: 99%

“…Parameters influencing liposome binding and nucleic acid delivery such as pH, composition of the osmoticum, Ca2+ ions, and polyethylene glycol have been studied (4,9,12,(14)(15)(16)20).…”

mentioning

confidence: 99%

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Targeting of Large Liposomes with Lectins Increases Their Binding to Plant Protoplasts

Sheehy

Lurquin²

1983

Plant Physiol.

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Soybean agglutinin, peanut agglutinia, and concanavalin A were covalently bound by condensation reaction to gangliosides and ceramides incorporated within the bilayer of multilameliar and unilameilar liposomes. These modMied Uiposomes had a much higher affinity for carrot and tobacco protoplasts except when concanavalin A was used.In addition, soybean aggutinin and concanavalin A were attached by ligand-specific binding to Uposomes containig cholesterol molecules derivatized with each lectin-specific sugar. This procedure allowed efficient crosslinking of Uposomes to protoplasts. The same effect was achieved with soybean agglutinin and peanut agglutinin when derivatized cholesterol was replaced by gangliosides. The implications of these findings for the Uposome-mediated nucleic acid transfer into protoplasts are discussed.

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Section: Discussionmentioning

confidence: 82%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Parameters influencing liposome binding and nucleic acid delivery such as pH, composition of the osmoticum, Ca2+ ions, and polyethylene glycol have been studied (4,9,12,(14)(15)(16)20).…”

mentioning

confidence: 99%

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Targeting of Large Liposomes with Lectins Increases Their Binding to Plant Protoplasts

Sheehy

Lurquin²

1983

Plant Physiol.

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“…Although it is still not clear, fusion with plasmalemma (19,20) and endocytosis of liposomes by protoplasts have both been demonstrated. It is the demonstration of the endocytosis which has prompted us to look into the possible use ofpH-sensitive liposomes for an improved liposomemediated delivery in higher plant protoplasts.…”

mentioning

confidence: 99%

Improved Cytoplasmic Delivery to Plant Protoplasts via pH-Sensitive Liposomes

Wang¹,

Hughes²,

Huang³

1986

Plant Physiol.

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We demonstrated that the liposomes composed of dioleolylphosphatidylethanolamine/cholesterol/oleic acid (4:4:2) dramatically release their contents at a pH of less than or equal to 6.0 and are capable of delivering their contents into the cytoplasm of higher plant protoplasts. This is shown by using a soluble fluorescent dye, calcein, as a liposome-entrapped marker. We found that calcein fluorescence was evenly distributed in the cytoplasm of wild carrot protoplasts after the incubation of protoplasts with liposomes in the presence of polyethylene glycol 6000. At 0.45 micro mole phospholipid per 6 x 105 protoplast, for example, the percentage of protoplasts which took up liposomes was 89% which was much higher than that achieved by conventional pH-insensitive liposomes. In this study, liposomes were prepared by a detergent dialysis method which avoided sonication and organic solvents. Thus macromolecules such as proteins and nucleic acids could be entrapped in the liposomes and delivered to the cytoplasm of the protoplasts.Liposomes have been used as a vehicle to deliver biologically active molecules into animal cells (24,29); however, the use of liposomes with higher plant cells is still being developed. Recently, liposomes have been used to deliver RNA or DNA to plant protoplasts. These studies used either the Ca-EDTA chelation method or the modified reverse-phase evaporation method to produce liposomes (7,8). The frequencies of transformation with liposome-mediated nucleic acid transfer were fairly low (10-6 -10-5) when liposomes were prepared by both Ca-EDTA chelation and REV2 method; however, compared with the calcium phosphate coprecipitation method for transformation, liposome entrapment is capable of keeping the nucleic acid from the nuclease attack. (28). The lipid suspension was sonicated with a bath sonicator (Laboratory Supplies, Hicksville, NY) and the pH was adjusted to 8. After the small unilamellar vesicles (SUV) were formed by sonication, octylglucoside and calcein were added and the lipid suspension was transferred to a dialysis bag. Octylglucose was chosen because it has a high critical micellar concentration, which facilitates rapid removal from the mixed micelle complex (13). The molar ratio of octylglucoside to lipid used in this study was 10. The optimal condition for the production of vesicles occurred when the detergent/lipid ratio was above 10:1 (23). The concentration of calcein was 23.5 mM in the mixture of a final volume of 0.34 ml. After the mixture was dialyzed against 100 ml of Tris buffer (pH 8.0) containing 1 mm EDTA, 183.3 mm D-glucose, 23.5 mM calcein and 1 g washed SM-2 beads (14) for 24 h, the liposomes were extruded through a polycarbonate filter of 0.2 ,um pore diameter (Nuclepore Corp.) to form vesicles of a uniform size distribution. Subsequently, the liposomes were separated from free calcein using a column of autoclaved Sepharose CL-2B.

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Plant Genetic Engineering Via Organelle Transfer

McDaniel

1984

Plant Breeding Reviews

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Binding of large liposomes to plant protoplasts and delivery of encapsulated DNA

Cited by 29 publications

References 20 publications

Targeting of Large Liposomes with Lectins Increases Their Binding to Plant Protoplasts

Targeting of Large Liposomes with Lectins Increases Their Binding to Plant Protoplasts

Improved Cytoplasmic Delivery to Plant Protoplasts via pH-Sensitive Liposomes

Plant Genetic Engineering Via Organelle Transfer

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