Search

To fully understand the function of cytomegalovirus (CMV) genes, it is imperative that they be studied in the context of infection. Therefore, the targeted deletion of individual viral genes and the comparison of loss of function viral mutants to the wild-type virus allow the identification of the relevance and role for a particular gene in the viral replication cycle. Targeted CMV mutagenesis has made huge advances over the past 15 years. The cloning of CMV genomes into (E. coli) as bacterial artificial chromosomes (BAC) allows not only quick and efficient deletion of viral genomic regions, individual genes, or single nucleotide exchanges in the viral genome but also the insertion of heterologous genetic sequences for gain of function approaches. The conceptual advantage of this strategy is that it overcomes the restrictions of recombinant technologies in cell culture systems. Namely, recombination in infected cells occurs only in a few clones, and their selection is not possible if the targeted genes are relevant for virus replication and are not able to compete for growth against the unrecombined viruses. On the other hand, BAC mutagenesis enables the selection for antibiotic resistance in E. coli, allowing a selective growth advantage to the recombined genomes. Here we describe the methods used for the generation of a CMV BAC, targeted mutagenesis of BAC clones, and transfection of human cells with CMV BAC DNA in order to reconstitute the viral infection process. [ABSTRACT FROM AUTHOR]

We describe simple and efficient plastid transformation methods for suspension-cultured cells and sporelings of the liverwort, Marchantia polymorpha L. Use of rapidly proliferating cells such as suspension-cultured cells and sporelings, which are immature thalli developing from spores, as targets made plastid transformation by particle bombardment efficient. Selection on a sucrose-free medium and linearization of the transformation vector significantly improved the recovery rate of plastid transformants. With the methods described here, a few plastid transformants are obtained from a single bombardment of sporelings, while more efficient plastid transformation is expected in suspension-cultured cells, ~60 transformants from a single bombardment. Homoplasmic transformants of thalli are obtained immediately after primary selection, whereas homoplasmic transformants from suspension-cultured cells are obtained after 12–16 weeks of repeated subculture. [ABSTRACT FROM AUTHOR]

The moss Physcomitrella patens performs efficient homologous recombination in both the nucleus and plastid enabling the study of individual gene function by generating precise inactivation or modification of genes. Polyethylene glycol (PEG)-mediated transformation of protoplasts is routinely used to study the nuclear gene function of P. patens. PEG-mediated protoplast transformation is also applied for plastid transformation of this moss. The efficiency of plastid transformation is quite reliable, and one or two homoplasmic transplastomic lines are obtained in a plastid transformation experiment (5 × 105 protoplasts) by selection for spectinomycin resistance. [ABSTRACT FROM AUTHOR]

The availability of routine techniques for the genetic manipulation of the chloroplast genome of Chlamydomonas reinhardtii has allowed a plethora of reverse-genetic studies of chloroplast biology using this alga as a model organism. These studies range from fundamental investigations of chloroplast gene function and regulation to sophisticated metabolic engineering programs and to the development of the algal chloroplast as a platform for producing high-value recombinant proteins. The established method for delivering transforming DNA into the Chlamydomonas chloroplast involves microparticle bombardment, with the selection of transformant lines most commonly involving the use of antibiotic resistance markers. In this chapter we describe a simpler and cheaper delivery method in which cell/DNA suspensions are agitated with glass beads: a method that is more commonly used for nuclear transformation of Chlamydomonas. Furthermore, we highlight the use of an expression vector (pASapI) that employs an endogenous gene as a selectable marker, thereby avoiding the contentious issue of antibiotic resistance determinants in transgenic lines. [ABSTRACT FROM AUTHOR]

Although plastid transformation has attractive advantages and potential applications in plant biotechnology, for long time it has been highly efficient only in tobacco. The lack of efficient selection and regeneration protocols and, for some species, the inefficient recombination using heterologous flanking regions in transformation vectors prevented the extension of the technology to major crops. However, the availability of this technology for species other than tobacco could offer new possibilities in plant breeding, such as resistance management or improvement of nutritional value, with no or limited environmental concerns. Herein we describe an efficient plastid transformation protocol for potato (Solanum tuberosum subsp. tuberosum). By optimizing the tissue culture system and using transformation vectors carrying homologous potato flanking sequences, we obtained up to one transplastomic shoot per bombardment. Such efficiency is comparable to that usually achieved in tobacco. The method described in this chapter can be used to regenerate potato transplastomic plants expressing recombinant proteins in chloroplasts as well as in amyloplasts. [ABSTRACT FROM AUTHOR]

Excision of marker genes using DNA direct repeats makes use of the predominant homologous recombination pathways present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and microalgae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required, and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes. Release of selection allows the accumulation of marker-free plastid genomes generated by marker excision, which is spontaneous, random, and a unidirectional process. Positive selection is provided by linking marker excision to restoration of the coding region of an herbicide resistance gene from two overlapping but incomplete coding regions. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat-mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastid genomes during growth, development and flowering of T0 plants allows the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops. [ABSTRACT FROM AUTHOR]

We have explained some experimental data of the homologous recombination and the genetic interference in terms of one-dimensional random walk over discrete sites. We first review our previous results. Next, we modify our random-walk model for the homologous recombination into a continuous-site model, and discuss a possible explanation for the previous experimental data obtained by means of the plasmid having one-side homology. Finally, we show that a reaction between an intermediate and a product is indispensable in explaining the genetic interference in terms of our reaction-diffusion model. [ABSTRACT FROM AUTHOR]

Having many characteristics of an ideal experimental system, Arabidopsis thaliana became a very important model system for flowering plants. Its completed genome sequence data provided scientists the first fundamental tool towards understanding its genome structure and genes that it possess. There are more than 33,000 predicted genes in Arabidopsis and this number is increasing as novel methods develop and our understanding of genome organization and regulation expands. Reverse genetics that aim to reveal the functions of all Arabidopsis genes and the related resources were next most important tools that plant scientists needed. Numerous consortia were formed to supply scientists with such resources and tools necessary to determine the functions of Arabidopsis genes. Thanks to these international community efforts, now there are around 426,000 independent T-DNA/transposon insertion lines representing near saturation of all genes in Arabidopsis available for the research community. Besides insertion lines, several other sophisticated technologies and resources crucial for large-scale gene function studies in Arabidopsis were also developed. In this chapter, we discuss most of these important reverse genetics resources for gene function analysis. [ABSTRACT FROM AUTHOR]

The use of genetically manipulated mice in medical research is one of the premier tools for the study of genetic diseases. I describe here our routine methods to produce these animals that have proven to be highly reliable as well as give exceptionally high rates of germline transmission with all strains of embryonic stem cells that we have used. [ABSTRACT FROM AUTHOR]