scholarly journals A universal vector concept for a direct genotyping of transgenic organisms and a systematic creation of homozygous lines

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Frederic Strobl ◽  
Anita Anderl ◽  
Ernst HK Stelzer
Keyword(s):  
Tribolium Castaneum ◽  
Live Imaging ◽  
Model Organisms ◽  
Knock Out ◽  
Transgenic Organisms ◽  
Transgenic Organism ◽  
Transgenic Lines ◽  
Diploid Model

Diploid transgenic organisms are either hemi- or homozygous. Genetic assays are, therefore, required to identify the genotype. Our AGameOfClones vector concept uses two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to hemizygous individuals that carry only one marker. In the following generation, heterozygous descendants are identified by the presence of both markers and produce homozygous progeny that are selected by the lack of one marker. We prove our concept in Tribolium castaneum by systematically creating multiple functional homozygous transgenic lines suitable for long-term fluorescence live imaging. Our approach saves resources and simplifies transgenic organism handling. Since the concept relies on the universal Cre-Lox system, it is expected to work in all diploid model organisms, for example, insects, zebrafish, rodents and plants. With appropriate adaptions, it can be used in knock-out assays to preselect homozygous individuals and thus minimize the number of wasted animals.

scholarly journals The beetle amnion and serosa functionally interact as apposed epithelia

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Maarten Hilbrant ◽  
Thorsten Horn ◽  
Stefan Koelzer ◽  
Kristen A Panfilio
Keyword(s):  
Tribolium Castaneum ◽  
Live Imaging ◽  
Evolutionary Changes ◽  
Late Embryogenesis ◽  
Previous Hypothesis ◽  
Transgenic Lines

Unlike passive rupture of the human chorioamnion at birth, the insect extraembryonic (EE) tissues – the amnion and serosa – actively rupture and withdraw in late embryogenesis. Withdrawal is essential for development and has been a morphogenetic puzzle. Here, we use new fluorescent transgenic lines in the beetle Tribolium castaneum to show that the EE tissues dynamically form a basal-basal epithelial bilayer, contradicting the previous hypothesis of EE intercalation. We find that the EE tissues repeatedly detach and reattach throughout development and have distinct roles. Quantitative live imaging analyses show that the amnion initiates EE rupture in a specialized anterior-ventral cap. RNAi phenotypes demonstrate that the serosa contracts autonomously. Thus, apposition in a bilayer enables the amnion as 'initiator' to coordinate with the serosa as 'driver' to achieve withdrawal. This EE strategy may reflect evolutionary changes within the holometabolous insects and serves as a model to study interactions between developing epithelia.

scholarly journals Non-invasive long-term fluorescence live imaging of Tribolium castaneum embryos

Development ◽  
2014 ◽  
Vol 141 (11) ◽  
pp. 2361-2361 ◽  
Author(s):  
F. Strobl ◽  
E. H. K. Stelzer
Keyword(s):  
Tribolium Castaneum ◽  
Live Imaging ◽  
Non Invasive

scholarly journals The beetle amnion and serosa functionally interact as apposed epithelia

2015 ◽  
Author(s):  
Stefan Koelzer ◽  
Maarten Hilbrant ◽  
Thorsten Horn ◽  
Kristen A. Panfilio
Keyword(s):  
Tribolium Castaneum ◽  
Live Imaging ◽  
The Novel ◽  
Functional Interaction ◽  
Successful Development ◽  
Evolutionary Changes ◽  
Late Embryogenesis ◽  
Transgenic Lines ◽  
And Function ◽  
High Degree

Unlike passive rupture of the human chorioamnion at birth, the insect extraembryonic (EE) tissues – the amnion and serosa – actively rupture and withdraw in late embryogenesis. Despite its importance for successful development, EE morphogenesis remains poorly understood. Contradicting the hypothesis of a single, fused EE membrane, we show that both tissues persist as discrete epithelia within a bilayer, using new tissue-specific EGFP transgenic lines in the beetle Tribolium castaneum. Quantitative live imaging analyses show that the amnion initiates EE rupture in a specialized anterior-ventral cap, while RNAi manipulation of EE tissue complement and function reveals that the serosa is autonomously contractile. Thus the bilayer efficiently coordinates the amnion as initiator and serosa as driver to achieve withdrawal. The novel bilayer architecture may reflect evolutionary changes in the EE tissues specific to holometabolous insects. More generally, tissue apposition in a bilayer exemplifies a high degree of functional interaction between developing epithelia.

scholarly journals Non-invasive long-term fluorescence live imaging of Tribolium castaneum embryos

Development ◽  
2014 ◽  
Vol 141 (11) ◽  
pp. 2331-2338 ◽  
Author(s):  
F. Strobl ◽  
E. H. K. Stelzer
Keyword(s):  
Tribolium Castaneum ◽  
Live Imaging ◽  
Non Invasive

Pharmaceutical and Biotechnological Applications of Transgenic Technology

2018 ◽  
Vol 11 (4) ◽  
pp. 4145-4153
Author(s):  
D Samba Reddy ◽  
Tina Reddy
Keyword(s):  
Mammalian Species ◽  
Genetic Diseases ◽  
Transgenic Animals ◽  
Transgenic Animal ◽  
Pharmaceutical Products ◽  
Conditional Knockout ◽  
Knock Out ◽  
Transgenic Lines ◽  
Health And Disease ◽  
Or Genes

A transgenic animal is a genetically modified species in which researchers have modified an existing gene or genes by genetic engineering techniques. Genetic modification involves the mutation, insertion, or deletion of genes. Mouse is the most widely used mammalian species for creating transgenic lines. There are two types of transgenic animals: (i) gene deleted (“knock-out”) and (ii) gene overexpressed (“knock-in”). The loss or gain of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior and other observable characteristics. Knockout mice are key animal models for studying the role of genes which have been sequenced but whose functions have not been determined.  They include constitutive knockouts (gene deleted since birth) and conditional knockout (gene turned off later after birth).  The first knockout mouse was created in 1989 by Mario Capecchi, Martin Evans, and Oliver Smithies, for which they were awarded the 2007 Nobel Prize in Physiology or Medicine.  Transgenic mouse models have revolutionized the biomedical research and provided a power tool for understanding health and disease. Transgenic animals have been created for bulk production of biotechnology and pharmaceutical products.  In 2009, the FDA approved the first human biological drug ATryn, an anticoagulant extracted from the transgenic goat's milk. The recently discovered CRISPER gene editing technology is providing new frontiers in correcting abnormal genes and hopefully provide cures for genetic diseases in the future.    

scholarly journals Efficient Cryopreservation of Populus tremula by In Vitro-Grown Axillary Buds and Genetic Stability of Recovered Plants

Plants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 77
Author(s):  
Elena O. Vidyagina ◽  
Nikolay N. Kharchenko ◽  
Konstantin A. Shestibratov
Keyword(s):  
Survival Rate ◽  
Axillary Buds ◽  
Long Term Storage ◽  
Average Survival ◽  
Transgenic Lines ◽  
Ssr Loci ◽  
One Step ◽  
The One

Axillary buds of in vitro microshoots were successfully frozen at –196 °C by the one-step freezing method using the protective vitrification solution 2 (PVS2). Microshoots were taken from 11 transgenic lines and three wild type lines. Influence of different explant pretreatments were analyzed from the point of their influence towards recovery after cryopreservation. It was found out that the use of axillary buds as explants after removal of the apical one increases recovery on average by 8%. The cultivation on growth medium of higher density insignificantly raises the regenerants survival rate. Pretreatment of the osmotic fluid (OF) shows the greatest influence on the survival rate. It leads to the increase in survival rate by 20%. The cryopreservation technology providing regenerants average survival rate of 83% was developed. It was based on the experimental results obtained with explant pretreatment. Incubation time in liquid nitrogen did not affect the explants survival rate after thawing. After six months cryostorage of samples their genetic variability was analyzed. Six variable simple sequence repeat (SSR) loci were used to analyze genotype variability after the freezing-thawing procedure. The microsatellite analysis showed the genetic status identity of plants after cryopreservation and of the original genotypes. The presence of the recombinant gene in the transgenic lines after cryostorage were confirmed so as the interclonal variation in the growth rate under greenhouse conditions. The developed technique is recommended for long-term storage of various breeding and genetically modified lines of aspen plants, as it provides a high percentage of explants survival with no changes in genotype.

scholarly journals Transgenic Epigenetics: Using Transgenic Organisms to Examine Epigenetic Phenomena

2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
Lori A. McEachern
Keyword(s):  
Molecular Mechanisms ◽  
Model Organism ◽  
Evolutionary Conservation ◽  
Model Organisms ◽  
Valuable Insight ◽  
Epigenetic Control ◽  
Transgenic Organisms ◽  
Epigenetic Analysis ◽  
Insight Into ◽  
Epigenetic Processes

Non-model organisms are generally more difficult and/or time consuming to work with than model organisms. In addition, epigenetic analysis of model organisms is facilitated by well-established protocols, and commercially-available reagents and kits that may not be available for, or previously tested on, non-model organisms. Given the evolutionary conservation and widespread nature of many epigenetic mechanisms, a powerful method to analyze epigenetic phenomena from non-model organisms would be to use transgenic model organisms containing an epigenetic region of interest from the non-model. Interestingly, while transgenic Drosophila and mice have provided significant insight into the molecular mechanisms and evolutionary conservation of the epigenetic processes that target epigenetic control regions in other model organisms, this method has so far been under-exploited for non-model organism epigenetic analysis. This paper details several experiments that have examined the epigenetic processes of genomic imprinting and paramutation, by transferring an epigenetic control region from one model organism to another. These cross-species experiments demonstrate that valuable insight into both the molecular mechanisms and evolutionary conservation of epigenetic processes may be obtained via transgenic experiments, which can then be used to guide further investigations and experiments in the species of interest.

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