We investigated the size of flanking DNA incorporated into the tobacco plastid genome alongside a selectable antibiotic resistance mutation. The results showed that integration of a long uninterrupted region of homologous DNA, rather than of small fragments as previously thought, is the more likely event in plastid transformation of land plants. Transforming plasmid pJS75 contains a 6.2-kb DNA ...

Methods pPRV111A::neoSTLS2 construction and transplastomic plants pPRV111A::neoSTLS2 was produced by ligating the HindIII fragment containing neoSTLS2 from plasmid pCMneoSTLS2 (ref. 13) into pPRV111A. This transformation vector was introduced into the plastids of Nicotiana tabacum L. Petit Havana (N,N) by biolistic bombardment, and homoplasmic plants were regenerated as described previously. Th...

Biolistic gun-mediated transformation is one of the two most effective and popular methods for introducing genes into maize. In this chapter, we describe a detailed protocol for genetic transformation of the maize genotype, Hi II. The protocol uses 0.6-microm gold particles as microcarriers and the herbicide resistance bar gene as a selective marker. Both immature zygotic embryos and immature e...

Our understanding of G protein-coupled receptors (GPCRs) in the central nervous system (CNS) has been hampered by the limited availability of tools allowing for the study of their signaling with precise temporal control. To overcome this, we tested the utility of the bistable mammalian opsin melanopsin to examine G protein signaling in CNS neurons. Specifically, we used biolistic (gene gun) app...

Our aim is the biolistic transfection of organotypic cultures of rat visual cortex with plasmids coding for neurotrophic factors, which then become expressed for limited periods of time during postnatal ontogenesis. Out of two commercially available devices, we adopted the handheld 'Helios Gene Gun' instead of the stationary PDS-1000He (both Biorad, Munich, Germany). This device allows multiple...

The delivery of proteins instead of DNA into plant cells allows for a transient presence of the protein or enzyme that can be useful for biochemical analysis or genome modifications. This may be of particular interest for genome editing, because it can avoid DNA (transgene) integration into the genome and generate precisely modified "nontransgenic" plants. In this work, we explore direct protei...

Here we report preparation and properties of positively charged gold microparticles, and their use for biolistic DNA delivery. Micron-sized gold microparticles were modified by building self-assembling polyethylenimine monolayers on their surfaces, which enabled their electrostatic interaction with negatively charged molecules such as DNA. One milligram of the surface-modified microparticles wa...

Ion channels and PSD-95 are colocalized in specific neuronal subcellular locations by an unknown mechanism. To investigate mechanisms of localization, we used biolistic techniques to express GFP-tagged PSD-95 (PSD-95:GFP) and the K(+)-selective channel Kv1.4 in slices of rat cortex. In pyramidal cells, PSD-95:GFP required a single PDZ domain and a region including the SH3 domain for localizatio...