Volume 125, number 2 FEBS LETTERS March 1981 zyxwvutsr QUANTITATIVE ASPECTS OF NUCLEIC ACIDS SEQUESTRATION IN LARGE LIPOSOMES AND THEIR EFFECTS ON PLANT PROTOPLASTS Paul F. LURQUIN, Raymond E. SHEEHY and Nancy A. RAO progvarn zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA in G3v3ics (kfokcdar Genetics, Heald Hall), Washington State University, Pullnlan, WA 99164, USA Received 23 January 1981 1. Introduction Large liposomes have been shown to entrap RNA and DNA molecules ([l-5] and references therein). When incubated with living cells, nucleic acids seques- tered in lipid vesicles were also shown to be efficiently transferred into cellular compartments where they became functionally expressed. For instance, polio- virus RNA included in LG-phosphatidyl-L-serine (PS) large unilamellar liposomes (LUV) transfected mammalian cells at high frequency [I]. Similarly, the Escherichia colt’ p-lactamase gene isolated from the plasmid pBR322 and encapsulated in PS small uni- lamellar liposomes was shown to be incorporated and apparently expressed in mammalian cells [2]. Thus, liposomes seem to constitute promising vectors for the genetic engineering of animal cells. Although transformation in plant cells still awaits final proof, it appears that here also, liposomes have considerable potential in achieving this goal. Indeed, previous studies suggest that DNA sequestered in large liposomes can under certain conditions be trans- ferred to plant protoplasts nuclei in a rather unde- graded form [4-81. However, DNA uptake by higher plant protoplasts presents specific problems [4,5] and hence, several parameters regarding, i.e., DNA trapping efficiency in liposomes and lipid cytotoxicity must be determined in order to promote high DNA transfer values in the absence of major cellular damage. This paper describes the sequestration of linear and cova- lently closed circular DNA and single-stranded plant viral RNA in different types of liposomes. Some of their effects on cowpea and carrot protoplasts are also investigated. ElsellierlNorth-Holland Biomedical Press 2. Materials and methods 2.1. Nucleic acids Cowpea chlorotic mottle virus (CCMV) RNA was isolated as in [1 l] and was a gift from Dr S. Wyatt (Department of Plant Pathology, WSU). Co1 Er [3H]DNA (800 cpm/mg) was isolated from E. cofi JC 411 after chloramphenicol amplification and was a gift from R. Calza (Program in Genetics, WSU). Low sequestration values obtained with PS-LUV and PS multilamellar vesicles (MLV) were confirmed by using [“HI DNA of 1O-fold higher specific radioactivity. Agrobacterium tumefacierzs C58 pTi [3H] DNA (1000 cpm/mg) was a gift from Dr F. van Vliet (Genet- ics Laboratory, State University of Ghent). R6K, pCR1 and pBR322 plasmid DNAs were isolated as in [lo]. Salmon sperm DNA (Sigma) needle-sheared to nil, 5 X lo6 was complexed with ethidium bromide (EtBr) as in [4] prior to encapsulation in liposomes. 2.2. Liposomes Neutral MLV composed of L-o-phosphatidyl choline (lecithin) (Sigma, type V-E from egg yolk) were produced by mechanical shaking as in [4] except that shaking of the lipid film in the presence of DNA dissolved in ‘liposome buffer’ [4] was reduced to 15 s. Negatively charged MLV composed of L+phospha- tidyl-L-serine (Sigma) or lecithin plus increasing amounts of dicetyl phosphate (Sigma) were similarly produced. Positively charged MLV composed of lecithin and increasing amounts of stearylamine (Sigma) were also obtained by mechanical shaking. Positively charged LUV of identical composition were obtained by ether infusion as in [5]. Negatively charged PS-LUV were generated from sonicated lipo- somes as in [9]. All liposomes were spun down at 183