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Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins
Background
Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient.
Results
Here we describe
Easi-
CRISPR (
E
fficient
a
dditions with
s
sDNA
i
nserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that
Easi
-CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring.
Conclusions
Easi-
CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus,
Easi-
CRISPR offers a comprehensive means of building large-scale Cre-
LoxP
animal resources.
Journal Article
Legitimation in a world at risk : the case of genetically modified crops in India
This book provides a sociological analysis of the controversy surrounding GM crops in Telangana, India. There is much debate as to whether GM technology holds the key to improving the welfare of poor farmers globally or serves primarily to increase the profits of multinational corporations while enhancing cultivator risk. Desmond's study is located in the economically vulnerable and politically volatile district of Warangal in Telangana, a context associated with high numbers of farmer suicides. Uniquely foregrounding the perspectives of cultivators and the landless, Desmond explores how GM crops are variously legitimated and delegitimated in three Warangal villages by those whose livelihoods are at stake in the debate, but whose voices are rarely heard within it. This book will be significant for those with an interest in GM crops, power and knowledge and their relation to understandings of development, democracy and risk management worldwide.
Book
Brain tyrosinase overexpression implicates age-dependent neuromelanin production in Parkinson’s disease pathogenesis
In Parkinson’s disease (PD) there is a selective degeneration of neuromelanin-containing neurons, especially substantia nigra dopaminergic neurons. In humans, neuromelanin accumulates with age, the latter being the main risk factor for PD. The contribution of neuromelanin to PD pathogenesis remains unknown because, unlike humans, common laboratory animals lack neuromelanin. Synthesis of peripheral melanins is mediated by tyrosinase, an enzyme also present at low levels in the brain. Here we report that overexpression of human tyrosinase in rat substantia nigra results in age-dependent production of human-like neuromelanin within nigral dopaminergic neurons, up to levels reached in elderly humans. In these animals, intracellular neuromelanin accumulation above a specific threshold is associated to an age-dependent PD phenotype, including hypokinesia, Lewy body-like formation and nigrostriatal neurodegeneration. Enhancing lysosomal proteostasis reduces intracellular neuromelanin and prevents neurodegeneration in tyrosinase-overexpressing animals. Our results suggest that intracellular neuromelanin levels may set the threshold for the initiation of PD.
It is unclear if neuromelanin plays a role in Parkinson’s disease pathogenesis since common laboratory animals lack this pigment. Authors show here that overexpression of human tyrosinase in the substantia nigra of rats resulted in an age-dependent production of human-like neuromelanin within nigral dopaminergic neurons and is associated with a Parkinson’s disease phenotype when allowed to accumulate above a specific threshold.
Journal Article
Corporate crops : biotechnology, agriculture, and the struggle for control
Frontmatter -- Contents -- List of Acronyms -- Introduction -- 1 Agricultural Biotechnologies on the Farm and around the World -- 2 The Coming of the Third Regime? Agricultural Biotechnology Regulation in Canada and the United States -- 3 Biotechnology on the Prairies: The Rise of Canola . . . -- 4 . . . And the Fall of Wheat -- 5 Legal Offense and Defense on the Canadian Prairies -- 6 From When Cotton Was King to King Monsanto -- 7 Starting a New Regime: Training the Locals -- 8 Conclusion -- Appendix: Log of Interviews -- Notes -- Bibliography -- Index
eBook
We are not starving : the struggle for food sovereignty in Ghana
\"We Are Not Starving is an ethnography of how global powers, local resistance, and capital flows are shaping contemporary African foodways\"--Provided by publisher.
BOOK
OsMADS51 Is a Short-Day Flowering Promoter That Functions Upstream of Ehd1, OsMADS14, and Hd3a1WOA
Although flowering regulatory mechanisms have been extensively studied in Arabidopsis (Arabidopsis thaliana), those in other species have not been well elucidated. Here, we investigated the role of OsMADS51, a type I MADS-box gene in the short-day (SD) promotion pathway in rice (Oryza sativa). In SDs OsMADS51 null mutants flowered 2 weeks later than normal, whereas in long days loss of OsMADS51 had little effect on flowering. Transcript levels of three flowering regulators-Ehd1, OsMADS14, and Hd3a-were decreased in these mutants, whereas those of OsGI and Hd1 were unchanged. Ectopic expression of OsMADS51 caused flowering to occur about 7 d earlier only in SDs. In ectopic expression lines, transcript levels of Ehd1, OsMADS14, and Hd3a were increased, but those of OsGI and Hd1 remained the same. These results indicate that OsMADS51 is a flowering promoter, particularly in SDs, and that this gene functions upstream of Ehd1, OsMADS14, and Hd3a. To further investigate the relationship with other flowering promoters, we generated transgenic plants in which expression of Ehd1 or OsGI was suppressed. In Ehd1 RNA interference plants, OsMADS51 expression was not affected, supporting our conclusion that the MADS-box gene functions upstream of Ehd1. However, in OsGI antisense plants, the OsMADS51 transcript level was reduced. In addition, the circadian expression pattern for this MADS-box gene was similar to that for OsGI. These results demonstrate that OsMADS51 functions downstream of OsGI. In summary, OsMADS51 is a novel flowering promoter that transmits a SD promotion signal from OsGI to Ehd1.
Journal Article
Correction: Monitoring of Cd and GSH contents and Bn-OASTL expression in transgenic tobacco seedlings in response to Cd stress
[This corrects the article DOI: 10.1371/journal.pone.0329885.].
Journal Article