Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker.

Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms. © 2011 The Author(s)

Status of clean gene (selection marker-free)technology

The ever-increasing world population has created two major problems: more mouths to feed and less land to farm. While classical breeding has enormously helped in providing more food, a lot still needsto be done. Transformation biotechnology can also help especially where classical breeding lacks solution (e.g. limited availability of stable and durable genetic source of resistance). However, planttransformation technologies require selectable marker genes to produce transgenic plants but such genes are of no value thereafter; in fact, marker genes in transgenic plants are perceived to pose potential bio-safety problems. In all genetic transformation technologies based on direct gene transfer (electroporation of protoplasts, particle bombardment etc) the selectable marker genes generally cointegratewith the gene of interest(s) in one Mendelian locus in the plant genome; hence, their removal is highly desirable. This may also help in the acceptability of transgenic plants by society. Transgenicplants that contain the desired gene of interest but lack the selection marker gene used in its production are termed “clean” and the methods utilized in their production are referred to as “clean gene” technology. There are several proved methods of eliminating selectable marker genes and these include: (A) Marker gene excision consisting of (1) intra-genomic relocation of transgenes via transposable elements, (2) site-specific recombination systems and (3) intra-chromosomalrecombination. (B) Gene replacement or Targeted gene replacement; and (C) transformation with multiple T-DNAs, which could result in linked and/or unlinked co-integration of transgenes. Unlinkedtransgenes are then segregated out during meiosis

Methods to create a stringent selection system for mammalian cell lines

The efficient establishment of high protein producing recombinant mammalian cell lines is facilitated by the use of a stringent selection system. Here, we describe two methods to create a stringent selection system based on the Zeocin resistance marker. First, we cloned increasingly longer stretches of DNA, encoding a range of 8–131 amino acids immediately upstream of the Zeocin selection marker gene. The DNA stretches were separated from the open reading frame of the selection marker gene by a stopcodon. The idea behind this was that the translation machinery will first translate the small peptide, stop and then restart at the AUG of the Zeocin marker. This process, however, will become less efficient with increasingly longer stretches of DNA upstream of the Zeocin marker that has to be translated first. This would result in lower levels of the Zeocin selection marker protein and thus a higher selection stringency of the system. Secondly, we performed a genetic screen to identify PCR induced mutations in the Zeocin selection protein that functionally impair the selection marker protein. Both the insertion of increasingly longer peptides and several Zeocin selection protein mutants resulted in a decreasing number of stably transfected colonies that concomitantly displayed higher protein expression levels. When the Zeocin mutants were combined with very short small peptides (8–14 amino acids long), this created a flexible, high stringency selection system. The system allows the rapid establishment of few, but high protein producing mammalian cell lines

Anthocyanin production as a potential visual selection marker during plant transformation

A mutant allele of the transcription factor gene MYB10 from apple induces anthocyanin production throughout the plant. This gene, including its upstream promoter, gene coding region and terminator sequence, was introduced into apple, strawberry and potato plants to determine whether it could be used as a visible selectable marker for plant transformation as an alternative to chemically selectable markers, such as kanamycin resistance. After transformation, red coloured calli, red shoots and red well-growing plants were scored. Red and green shoots were harvested from apple explants and examined for the presence of the MYB10 gene by PCR analysis. Red shoots of apple explants always contained the MYB10 gene but not all MYB10 containing shoots were red. Strawberry plants transformed with the MYB10 gene showed anthocyanin accumulation in leaves and roots. No visible accumulation of anthocyanin could be observed in potato plants grown in vitro, even the ones carrying the MYB10 gene. However, acid methanol extracts of potato shoots or roots carrying the MYB10 gene contained up to four times higher anthocyanin content than control plants. Therefore anthocyanin production as result of the apple MYB10 gene can be used as a selectable marker for apple, strawberry and potato transformation, replacing kanamycin resistance

HisB as novel selection marker for gene targeting approaches in Aspergillus niger

Background For Aspergillus niger, a broad set of auxotrophic and dominant resistance markers is available. However, only few offer targeted modification of a gene of interest into or at a genomic locus of choice, which hampers functional genomics studies. We thus aimed to extend the available set by generating a histidine auxotrophic strain with a characterized hisB locus for targeted gene integration and deletion in A. niger. Results A histidine-auxotrophic strain was established via disruption of the A. niger hisB gene by using the counterselectable pyrG marker. After curing, a hisB - , pyrG - strain was obtained, which served as recipient strain for further studies. We show here that both hisB orthologs from A. nidulans and A. niger can be used to reestablish histidine prototrophy in this recipient strain. Whereas the hisB gene from A. nidulans was suitable for efficient gene targeting at different loci in A. niger, the hisB gene from A. niger allowed efficient integration of a Tet-on driven luciferase reporter construct at the endogenous non-functional hisB locus. Subsequent analysis of the luciferase activity revealed that the hisB locus is tight under non-inducing conditions and allows even higher luciferase expression levels compared to the pyrG integration locus. Conclusion Taken together, we provide here an alternative selection marker for A. niger, hisB, which allows efficient homologous integration rates as well as high expression levels which compare favorably to the well-established pyrG selection marker.EC/FP7/303864/EU/Bridging the world of fungi and dementia/PROFITSTU Berlin, Open-Access-Mittel - 201

W::Neo: A Novel Dual-Selection Marker for High Efficiency Gene Targeting in Drosophila

We have recently developed a so-called genomic engineering approach that allows for directed, efficient and versatile modifications of Drosophila genome by combining the homologous recombination (HR)-based gene targeting with site-specific DNA integration. In genomic engineering and several similar approaches, a “founder” knock-out line must be generated first through HR-based gene targeting, which can still be a potentially time and resource intensive process. To significantly improve the efficiency and success rate of HR-based gene targeting in Drosophila, we have generated a new dual-selection marker termed W::Neo, which is a direct fusion between proteins of eye color marker White (W) and neomycin resistance (Neo). In HR-based gene targeting experiments, mutants carrying W::Neo as the selection marker can be enriched as much as fifty times by taking advantage of the antibiotic selection in Drosophila larvae. We have successfully carried out three independent gene targeting experiments using the W::Neo to generate genomic engineering founder knock-out lines in Drosophila

Application of GFAT as a Novel Selection Marker to Mediate Gene Expression

The enzyme glutamine: fructose-6-phosphate aminotransferase (GFAT), also known as glucosamine synthase (GlmS), catalyzes the formation of glucosamine-6-phosphate from fructose-6-phosphate and is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway. For the first time, the GFAT gene was proven to possess a function as an effective selection marker for genetically modified (GM) microorganisms. This was shown by construction and analysis of two GFAT deficient strains, E. coli ΔglmS and S. pombe Δgfa1, and the ability of the GFAT encoding gene to mediate plasmid selection. The gfa1 gene of the fission yeast Schizosaccharomyces pombe was deleted by KanMX6-mediated gene disruption and the Cre-loxP marker removal system, and the glmS gene of Escherichia coli was deleted by using λ-Red mediated recombinase system. Both E. coli ΔglmS and S. pombe Δgfa1 could not grow normally in the media without addition of glucosamine. However, the deficiency was complemented by transforming the plasmids that expressed GFAT genes. The xylanase encoding gene, xynA2 from Thermomyces lanuginosus was successfully expressed and secreted by using GFAT as selection marker in S. pombe. Optimal glucosamine concentration for E. coli ΔglmS and S. pombe Δgfa1 growth was determined respectively. These findings provide an effective technique for the construction of GM bacteria without an antibiotic resistant marker, and the construction of GM yeasts to be applied to complex media

Development of cisgenic apples with durable resistance to apple scab

Most of the apple (Malus × domestica) growers are facing serious disease problems with apple scab which is caused by the fungus Venturia inaequalis. Developing a resistant variety in apple through classical breeding is very slow and inefficient. So, we aim at improving existing apple varieties through a new concept called “cisgenesis” which saves time and effort compared to classical breeding. Malus floribunda proved to be a good source of natural scab resistance genes. The genes HcrVf1 and HcrVf2, consisting of promoter, coding and terminator sequences in their natural configuration, were isolated from Malus floribunda and cloned into the binary vector pMF1. Apple cv. ‘Gala’ was transformed with pMF1 containing HcrVf1 and HcrVf2, individually or in combination. pMF1 can be used to obtain marker-free plants by recombinase-based excision of a fragment carrying undesired gene sequences, such as antibiotic-selection marker genes, leaving behind only the gene(s)-of-interest and one recombination site. Using this vector it is therefore possible to stack several genes by retransformation using the same selection procedure. In order to obtain durable resistance, we have the intention to combine different resistance genes from Malus either by stacking them one by one or by introducing them all together in one T-DNA. Performance of all different types of cisgenic plants will be evaluated by monitoring scab resistance levels phenotypically and by determining gene expression profiles through quantitative RT-PC

A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 1; peer review: 1 approved, 1 approved with reservations]

In the fission yeast Schizosaccharomyces pombe the prevailing approach for gene manipulations is based on homologous recombination of a PCR product that contains genomic target sequences and a selectable marker. The CRISPR/Cas9 system has recently been implemented in fission yeast, which allows for seamless genome editing without integration of a selection marker or leaving any other genomic ‘scars’. The published method involves manual design of the single guide RNA (sgRNA), and digestion of a large plasmid with a problematic restriction enzyme to clone the sgRNA. To increase the efficiency of this approach, we have established and optimized a PCR-based system to clone the sgRNA without restriction enzymes into a plasmid with a dominant natMX6 (nourseothricin) selection marker. We also provide a web-tool, CRISPR4P, to support the design of the sgRNAs and the primers required for the entire process of seamless DNA deletion. Moreover, we report the preparation of G1-synchronized and cryopreserved S. pombe cells, which greatly increases the efficiency and speed for transformations, and may also facilitate standard gene manipulations. Applying this optimized CRISPR/Cas9-based approach, we have successfully deleted over 80 different non-coding RNA genes, which are generally lowly expressed, and have inserted 7 point mutations in 4 different genomic regions