Henry Daniell: chloroplast genetic engineering.

(Bt) cry2Aa2 protein at46.1% in transgenic chloroplasts [1]. It was not only thehighest accumulation of protein in transgenic plants buta complete bacterial operon was successfully expressed,for the first time, resulting in the formation of stablecry2Aa2 crystals. Transgenes express large amounts offoreign protein due to the high copy number of the chloro-plast genome in each plant cell—up to 10,000 copies oftransgene per cell [2–8]. Transgenes are integrated intothe spacer regions of the chloroplast genome by homolo-gous recombination of flanking sequences. This allowssite-specific integration and eliminates concerns of posi-tion effect, frequently observed in nuclear transgenicplants. As a result, it is not necessary to screen large num-bers of putative transgenic lines to choose for high-levelexpression of transgenes. In contrast, all chloroplast trans-genic lines express similar levels of foreign proteins, with-in the range of physiological variations [9]. In addition,site-specific integration by homologous recombinationeliminates introduction of vector sequences, which isoften a concern in nuclear transformation achieved bynon-homologous recombination. Yet another advantageis the lack of transgene silencing in chloroplast transgenicplants, which is a serious concern in nuclear transforma-tion. There is no gene silencing in chloroplast transgeniclines at the transcriptional levels despite accumulation oftranscripts 169-fold higher than nuclear transgenic plants[10, 11]. Similarly, there is no post-transcriptional genesilencing despite accumulation of foreign proteins up to47% of the total plant protein in cp transgenic lines [1].In most angiosperms, plastids are maternally inherit-ed, which minimizes the concern of outcrossing of trans-genes [12, 13] and reduces the potential toxicity of trans-genic pollen to non-target insects [1]. Maternal inheri-tance of transgenes offers containment because of lack ofgene flow through pollen [13] and this has been demon-strated in different plant species [7, 12, 14]. The Danielllaboratory developed a chloroplast genome derived cyto-plasmic male sterility system in which the tapetal layersare destroyed in developing pollen, making transgenicplants male sterile without affecting other metabolic func-tions [15]. This provides yet another strategy for trans-gene containment. Chloroplast transgenic carrot plantshave been engineered to withstand salt concentrationsonly halophytes could tolerate [16]. The Daniell laboratoryhas conferred several other plant traits, including herbi-cide [12], insect [17], and disease [18] resistance, drought[10], phytoremediation [19] and production of biopolymers[20].The chloroplast is an ideal cellular location to expressand accumulate certain proteins or their biosyntheticproducts that would otherwise be harmful to the plant ifthey were expressed in the cytoplasm. This has beendemonstrated by the non-toxic effect of cholera toxin Bsubunit (CTB), a candidate oral subunit vaccine forcholera, when it was accumulated in large quantitieswithin transgenic plastids; in contrast, even very smallquantities of CTB were toxic when expressed in the cyto-plasm [9, 21]. Also, trehalose, which is a pharmaceuticalpreservative, was very toxic when it was accumulated inthe cytosol but was non-toxic when it was compartmen-talized within plastids [10].Oral delivery of vaccine antigens has been shown toyield high systemic IgG titers and high mucosal IgAtiters, enabling the immune system to fight germs at theirportals of entry. Therefore, the Daniell laboratory hasdemonstrated expression and assembly of several vac-cine antigens, including the cholera toxin B subunit (CTB)[9], the F1~V fusion antigen for plague [6]; the 2L21 pep-tide from the canine parvovirus (CPV) [21], the anthraxprotective antigen (PA) [22], and the NS3 protein as a vac-cine antigen for hepatitis C [6]. Cytotoxicity measure-