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Bahiagrass (Paspalum notatum var. saurae) is the predominant forage grass in Florida and in other subtropical regions. To improve pest resistance against fall armyworm [Spodoptera frugiperda (J. E. Smith)], an optimized cry1Fa gene encoding a [delta]-endotoxin from Bacillus thuringiensis was synthesized, subcloned under the transcriptional control of the constitutive ubi1 promoter, and introduced into the bahiagrass cultivar Tifton 9 by particle bombardment. Three transgenic bahiagrass lines were generated using minimal transgene expression cassettes without vector backbone. Southern blot analyses showed independent cry1Fa transgene integration patterns for the three lines. Transcripts of cry1Fa were detected in all three transgenic lines by reverse transcriptase polymerase chain reaction. Cry1Fa protein was detected in two lines by immuno-chromatography and quantitative Cry1Fa enzyme linked immunosorbent assay (ELISA). The Cry1Fa ELISA also indicated stable cry1Fa transgene expression in vegetative progeny plants of both lines. Cry1Fa expression levels correlated well to resistance levels determined by insect bioassays. An average mortality rate of 83% was observed when neonate larvae of fall armyworm were fed with transgenic leaves of the highest Cry1Fa expressing line. These results indicate that minimal expression cassette technology supports stable and high level expression of Cry1Fa in bahiagrass which can control fall armyworm, a devastating pest of forage grasses.

The systematic development of functional molecular markers requires a set of non-redundant ESTs. However large-scale EST sequencing projects are frequently based on random sequencing of shotgun cDNA clones, which usually results in high redundancy of ESTs. To increase the efficiency in the identification of novel ESTs/genes for the development of non-redundant markers, it is necessary to reduce the redundancy of sequencing by eliminating the redundant clones from the cDNA library. In order to reach this goal, we adopted the ‘total cDNA hybridization’ approach on a set of barley cDNAs from different libraries to remove redundant clones prior to sequencing. To quantify the accuracy of the approach we performed cluster analysis on the ESTs of the clones used in this study. The results show that up to 90% of the redundant clones that are present in clusters can be identified by this approach. Furthermore cluster analysis revealed that the set of non-hybridizing cDNA clones contains 82–89% ‘singletons’. Therefore, cDNA hybridization of arrayed colony filters represents an efficient and cheap procedure to deplete a cDNA library of redundant clones in order to generate non-redundant molecular markers. In addition, this approach is suitable for maximizing the rate of gene discovery in an EST programme. [Copyright &y& Elsevier]

Adeno-associated viruses (AAVs) have recently emerged as the leading vector for retinal gene therapy. However, AAV vectors which are capable of achieving clinically relevant levels of transgene expression and widespread retinal transduction are still an unmet need. Using rationally designed AAV2-based capsid variants, we investigate the role of capsid hydrophilicity and hydrophobicity as it relates to retinal transduction. We show that hydrophilic, single amino acid (aa) mutations (V387R, W502H, E530K, L583R) in AAV2 negatively impact retinal transduction when heparan sulfate proteoglycan (HSPG) binding remains intact. Conversely, addition of hydrophobic point mutations to an HSPG binding deficient capsid (AAV2ΔHS) lead to increased retinal transduction in both mouse and macaque. Our top performing vector, AAV2(4pMut)ΔHS, achieved robust rod and cone photoreceptor (PR) transduction in macaque, especially in the fovea, and demonstrates the ability to spread laterally beyond the borders of the subretinal injection (SRI) bleb. This study both evaluates biophysical properties of AAV capsids that influence retinal transduction, and assesses the transduction and tropism of a novel capsid variant in a clinically relevant animal model. Importance Rationally guided engineering of AAV capsids aims to create new generations of vectors with enhanced potential for human gene therapy. By applying rational design principles to AAV2-based capsids, we evaluated the influence of hydrophilic and hydrophobic amino acid (aa) mutations on retinal transduction as it relates to vector administration route. Through this approach we identified a largely deleterious relationship between hydrophilic aa mutations and canonical HSPG binding by AAV2-based capsids. Conversely, the inclusion of hydrophobic aa substitutions on a HSPG binding deficient capsid (AAV2ΔHS), generated a vector capable of robust rod and cone photoreceptor (PR) transduction. This vector AAV2(4pMut)ΔHS also demonstrates a remarkable ability to spread laterally beyond the initial subretinal injection (SRI) bleb, making it an ideal candidate for the treatment of retinal diseases which require a large area of transduction., (Copyright © 2021 American Society for Microbiology.)