Gene Transmission, Growth, and Exogeneous Growth Hormone Expression of G2 Transgenic Betta Fish (Betta imbellis)

Highlight ResearchThe F2 of GH-transgenic B. imbellis was successfully producedThe transgene inheritance by the F2 fish was more than 90%The growth and body size of transgenic fish was significantly higher than controlF2 fish reached a larger body size in a shorter period compared to the F1 AbstractIn our previous research, we had successfully produced G0 and G1 Pangasianodon hypophthalmus growth hormone (PhGH) transgenic B. imbellis, native ornamental betta from Indonesia, which its giant-sized variant has valuable price for the breeders. The G0 and G1 transgenic (TG) fish showed higher growth rate and body size compared to the non-transgenic (NT) fish. The study was aimed to produce and evaluate the consistencies of transgene transmission and expression in G2 generation. The growth rate and body size between TG and NT fish was also compared. The G2 generation was produced using crosses between TG and NT G1 fish: ♂TG × ♀TG, ♂TG × ♀NT, ♂NT × ♀TG, and ♂NT ×♀ NT. Fish were reared for 12 weeks, and transgene detection was performed using the polymerase chain reaction method (PCR) on isolated DNA from the caudal fin clips. The endogenous and exogenous GH expression analysis was conducted using the quantitative real-time PCR (qPCR) method. The results showed that the inheritance of the GH transgene by the G2 fish was more than 90% in all transgenic crosses. Endogenous GH was expressed at the same levels in the brain of TG and NT fish, but the exogenous GH expression was highly detected only in the TG fish. The G2 transgenic fish had a higher specific growth rate, up to 31%, compared to the control. The body length of TG crosses were 23−35% higher and had 111−135% higher body weight compared to NT fish. These results showed a promising approached in mass-producing stable lines of giant-sized betta using the GH-transgenic technology.

Challenges for monitoring (trans)gene-flow in the environment.

Monitoring the presence of transgenes in the environment depends on analytical detection methods and their measurement uncertainties. In this chapter, we aim to identify key methodological aspects and pinpoint the research bottlenecks and needs for building the capacity to effectively monitor transgene escape from genetically modified (GM) crops to wild relatives or landraces. We reviewed the iconic debate concerning the presence of transgenes in landraces of Mexican maize (with both positive and negative results for GM contamination shown over the years) to examine the impact of using different approaches to monitoring and detecting transgenes in landraces. Despite the lack of clear international guidelines that are specific for sampling and testing heterogeneous and dispersed landraces and wild relatives, transgene detection methods have developed significantly over the past decades. There is now an immensely valuable set of tools, approaches, and harmonized protocols that continue to develop and provide a way to evaluate transgene presence. To support this ongoing development and to steer it in directions that are particularly useful for addressing the challenges associated with detection in landraces and wild relatives, we offer lessons from our review for future work in this area particularly useful for addressing the challenges associated with detection in landraces and wild relatives, we offer lessons from our review for future work in this area.

Genetic engineering of sex chromosomes for batch cultivation of non-transgenic, sex-sorted males

The field performance of Sterile Insect Technique (SIT) is improved by sex-sorting and releasing only sterile males. This can be accomplished by resource-intensive separation of males from females by morphology. Alternatively, sex-ratio biasing genetic constructs can be used to selectively remove one sex without the need for manual or automated sorting, but the resulting genetically engineered (GE) control agents would be subject to additional governmental regulation. Here we describe and demonstrate a genetic method for the batch production of non-GE males. This method could be applied to generate the heterogametic sex (XY, or WZ) in any organism with chromosomal sex determination. We observed up to 100% sex-selection with batch cultures of more than 103 individuals. Using a stringent transgene detection assay, we demonstrate the potential of mass production of transgene free males.

CRISPR-Cas9 System for Plant Genome Editing: Current Approaches and Emerging Developments

Targeted genome editing using CRISPR-Cas9 has been widely adopted as a genetic engineering tool in various biological systems. This editing technology has been in the limelight due to its simplicity and versatility compared to other previously known genome editing platforms. Several modifications of this editing system have been established for adoption in a variety of plants, as well as for its improved efficiency and portability, bringing new opportunities for the development of transgene-free improved varieties of economically important crops. This review presents an overview of CRISPR-Cas9 and its application in plant genome editing. A catalog of the current and emerging approaches for the implementation of the system in plants is also presented with details on the existing gaps and limitations. Strategies for the establishment of the CRISPR-Cas9 molecular construct such as the selection of sgRNAs, PAM compatibility, choice of promoters, vector architecture, and multiplexing approaches are emphasized. Progress in the delivery and transgene detection methods, together with optimization approaches for improved on-target efficiency are also detailed in this review. The information laid out here will provide options useful for the effective and efficient exploitation of the system for plant genome editing and will serve as a baseline for further developments of the system. Future combinations and fine-tuning of the known parameters or factors that contribute to the editing efficiency, fidelity, and portability of CRISPR-Cas9 will indeed open avenues for new technological advancements of the system for targeted gene editing in plants.

Batch cultivation of non-transgenic males suitable for SIT programs

The field performance of Sterile Insect Technique (SIT) is improved by sex-sorting and releasing only the sterile males. This can be accomplished by resource-intensive separation of males from females by morphology. Alternatively, sex-ratio biasing genetic constructs can be used to selectively kill one sex without the need for manual or automated sorting, but the resulting genetically engineered (GE) control agents would be subject to additional governmental regulation. Here we describe and demonstrate a method for the batch production of non-GE males that is applicable for sex-selective production of males suitable for genetic biocontrol programs. This method could be applied to generate the heterogametic sex (XY, or WZ) in any organism with chromosomal sex determination. We observed up to 100% sex-selection with batch cultures of more than 103 individuals. Using a stringent transgene detection assay, we demonstrate the potential of mass rearing of transgene free males.