Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short. (C) 2009 Elsevier Inc. All rights reserved.

Improving taro (Colocasia esculenta var. esculenta) production using biotechnological approaches

Taro (Colocasia esculenta L. Schott) is an important crop worldwide but is of particular significance in many Pacific Island countries where it forms part of the staple diet and serves as an export commodity. Escalating pest and disease problems are jeopardizing taro production with serious implications to food security and trade. Biotechnological approaches to addressing pest and disease problems, such as somatic embryogenesis and transgenesis, are potentially viable options. However, despite biotechnological advancements in higher profile agronomic crops, such progress in relation to Colocasia esculenta var. esculenta has been slow. This paper reviews taro biology, highlights the cultural and economic significance of taro in Pacific Island countries and discusses the progress made towards the molecular breeding of this important crop to date.

Development of a transient, high-level expression platform for protein production in plants

Plants are an attractive alternative to conventional expression systems for the production of recombinant proteins and useful biologics, however, the economic viability of plant made proteins is strongly yield dependent. This study aimed to improve transgene expression levels in the plant host Nicotiana benthamiana using the Agroinfiltration transient expression platform. Independent investigation of the physical, chemical and genetic features associated with Agroinfiltration identified factors that improved transformation frequencies, elevated transgene expression levels and ultimately improved protein yield. The major outcome of this research was a novel hyper-expression system for biofarming recombinant proteins in plants.

Super DNAging - New insights into DNA integrity, genome stability and telomeres in the oldest old

Reductions in DNA integrity, genome stability, and telomere length are strongly associated with the aging process, age-related diseases as well as the age-related loss of muscle mass. However, in people reaching an age far beyond their statistical life expectancy the prevalence of diseases, such as cancer, cardiovascular disease, diabetes or dementia, is much lower compared to “averagely” aged humans. These inverse observations in nonagenarians (90–99 years), centenarians (100–109 years) and super-centenarians (110 years and older) require a closer look into dynamics underlying DNA damage within the oldest old of our society. Available data indicate improved DNA repair and antioxidant defense mechanisms in “super old” humans, which are comparable with much younger cohorts. Partly as a result of these enhanced endogenous repair and protective mechanisms, the oldest old humans appear to cope better with risk factors for DNA damage over their lifetime compared to subjects whose lifespan coincides with the statistical life expectancy. This model is supported by study results demonstrating superior chromosomal stability, telomere dynamics and DNA integrity in “successful agers”. There is also compelling evidence suggesting that life-style related factors including regular physical activity, a well-balanced diet and minimized psycho-social stress can reduce DNA damage and improve chromosomal stability. The most conclusive picture that emerges from reviewing the literature is that reaching “super old” age appears to be primarily determined by hereditary/genetic factors, while a healthy lifestyle additionally contributes to achieving the individual maximum lifespan in humans. More research is required in this rapidly growing population of super old people. In particular, there is need for more comprehensive investigations including short- and long-term lifestyle interventions as well as investigations focusing on the mechanisms causing DNA damage, mutations, and telomere shortening.

Mu in vitro Transposition Technology in Functional Genetics and Genomics: Applications on Mouse and Bacteriophages

Transposons are mobile elements of genetic material that are able to move in the genomes of their host organisms using a special form of recombination called transposition. Bacteriophage Mu was the first transposon for which a cell-free in vitro transposition reaction was developed. Subsequently, the reaction has been refined and the minimal Mu in vitro reaction is useful in the generation of comprehensive libraries of mutant DNA molecules that can be used in a variety of applications. To date, the functional genetics applications of Mu in vitro technology have been subjected to either plasmids or genomic regions and entire genomes of viruses cloned on specific vectors. This study expands the use of Mu in vitro transposition in functional genetics and genomics by describing novel methods applicable to the targeted transgenesis of mouse and the whole-genome analysis of bacteriophages. The methods described here are rapid, efficient, and easily applicable to a wide variety of organisms, demonstrating the potential of the Mu transposition technology in the functional analysis of genes and genomes. First, an easy-to-use, rapid strategy to generate construct for the targeted mutagenesis of mouse genes was developed. To test the strategy, a gene encoding a neuronal K+/Cl- cotransporter was mutagenised. After a highly efficient transpositional mutagenesis, the gene fragments mutagenised were cloned into a vector backbone and transferred into bacterial cells. These constructs were screened with PCR using an effective 3D matrix system. In addition to traditional knock-out constructs, the method developed yields hypomorphic alleles that lead into reduced expression of the target gene in transgenic mice and have since been used in a follow-up study. Moreover, a scheme is devised to rapidly produce conditional alleles from the constructs produced. Next, an efficient strategy for the whole-genome analysis of bacteriophages was developed based on the transpositional mutagenesis of uncloned, infective virus genomes and their subsequent transfer into susceptible host cells. Mutant viruses able to produce viable progeny were collected and their transposon integration sites determined to map genomic regions nonessential to the viral life cycle. This method, applied here to three very different bacteriophages, PRD1, ΦYeO3 12, and PM2, does not require the target genome to be cloned and is directly applicable to all DNA and RNA viruses that have infective genomes. The method developed yielded valuable novel information on the three bacteriophages studied and whole-genome data can be complemented with concomitant studies on individual genes. Moreover, end-modified transposons constructed for this study can be used to manipulate genomes devoid of suitable restriction sites.

Expression of microinjected foreign DNA in silkworm,bombyx mori

As a prelude to achieving transgenesis in Bombyx mori, conditions have been established for successful microinjection of cloned foreign genes into the silk worm eggs. A sharpened metallic needle is used to pierce the thick chorion layer of the eggsheil, approaching through a droplet of DNA solution deposited on its surface. The microinjection is carried out within 2-2.5 h after oviposition and the injected eggs show 3-5% hatchability and 80-90% survival. Such larvae continuously expressed the microinjected cloned reporter gene, beta-galactosidase, placed under the control of a constitutively expressed cytoplasmic actin A3 gene promoter from B. mori. The expression is seen in different tissues, viz. the fat body, tracheae and the silk glands, till the late larval instars. The microinjected DNA sequences are retained in the adult G(o) moths.

The review of recent advances in fish genetics and biotechnology

Great advances have been, and are being made in our knowledge of the genetics and molecular biology (including genomics, proteomics and structural biology). Global molecular profiling technologies such as microassays using DNA or oligonucleotide chip, and protein and lipid chips are being developed. The application of such biotechnological advances are inevitable in aquaculture in the areas of improvement of aquaculture stocks where many molecular markers such as RFLPs, AFLDs and RAPD are now available for genome analysis, finger printing and genetic linkage mapping. Transgenic technology has been developed in a number of fish species and research is being pursed to produce transgenic fish carrying genes that encode antimicrobial peptides such as lysozyme thereby achieving disease resistance in fish. Also it is a short cut to achieving genetic change for fast growth and other desirable traits like early sexual maturity, temperature tolerance and feed conversion efficiency. KEYWORDS: Fish genetics, transgenesis, monoploidy, diploidy, polyploidy,gynogenesis, androgenesis, cryopreservation.

Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites

With current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10(-4) to 10(-5)) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering.

Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

Clonación de gametas y transgénesis mediada por fertilización in-vitro en bovinos

En la presente tesis se desarrolló un método de clonación del genoma del espermatozoide y del ovocito bovino mediante la producción de embriones androgenéticos y partenogenéticos haploides. Esta técnica también fue utilizada para generar embriones bovinos que expresan un gen exógeno (transgen) en forma homogénea. Las tasas de desarrollo de los embriones reconstruídos utilizando genomas espermaticos clonados (blastomeras androgenéticas), alcanzaron 85.1 por ciento de clivaje, 9 por ciento de blastocistos y todos los embriones expresaron el transgen (EGFP) durante el desarrollo in-vitro. Las tasas de clivaje y de blastocistos de los embriones reconstruídos utilizando genomas clonados de ovocitos (blastomeras partenogenéticas), alcanzaron 78.4 por ciento y 10.8 por ciento respectivamente. Todos los embriones reconstruidos utilizando blastomeras partenogenéticas que expresaban el transgen mostraron expresión de EGFP y el 96.6 por ciento de ellos en forma homogénea. Posteriormente se desarrolló un nuevo método de transgenesis que permite transfectar cigotos de fertilización in vitro (FIV) y ovocitos activados partenogeneticamente (AP). El 70 por ciento de los embriones clivados y el 50 por ciento de los blastocistos expresaron EGFP cuando complejos pCX-EGFP-liposomas fueron inyectados 16 h post-fertilizacion y utilizando una concentración de 500 ng ADN exógeno.ƒÊl. Al inyectar ovocitos 3 h post-activación partenogénetica se obtuvo una tasa de expresión de 48.4 por ciento. Por otro lado, evaluamos la incidencia de fragmentación del ADN tras la inyección del transgen, demostrando que su expresión afecta la integridad del ADN en blastocistos bovinos de FIV, pero no así las tasas de desarrollo in vitro. En resumen, la presente tesis conforma una base sólida para concluir que es posible la clonación de genomas de ovocitos y espermatozoides con capacidad de generar embriones biparentales que evolucionan hasta estadio de blastocisto. Además, este procedimiento demostró ser una herramienta eficiente para la incorporación de genes exógenos en un embrión. Finalmente se demostró que la inyección intracitoplasmática de liposomas es una estrategia eficiente para introducir ADN exógeno en embriones de FIV y AP.