Okamura, E. | Search bioRxiv

bioRxiv2023

Highly efficient transgenic mouse production using piggyBac and its application to rapid phenotyping at the founder generation

Pronuclear microinjection is the most popular method for producing transgenic (Tg) animals. Because the production efficiency is typically less than 20%, phenotypic characterization of Tg animals is generally performed on the next generation (F1) onwards. However, apart from in rodents, in many animal species with long generation times, it is desirable to perform phenotyping in the founder (F0) generation. In this study, we attempted to optimize a method of Tg mouse production to achieve higher Tg production efficiency using piggyBac transposon systems and established optimal conditions under which almost all individuals in the F0 generation were Tg. We also succeeded in generating bacterial artificial chromosome Tg mice with efficiency of approximately 70%. By combining this method with genome editing technology, we established a new strategy to perform phenotyping of mice with tissue-specific knockout using the F0 generation. Taking the obtained findings together, by using this method, experimental research using Tg animals can be carried out more efficiently.

bioengineering↗

bioRxiv2018

No evidence for transvection in vivo by a superenhancer:promoter pair integrated into identical open chromatin at the Rosa26 locus

Long-range associations between enhancers and their target gene promoters have been shown to play critical roles in executing genome function. Recent variations of chromosome capture technology have revealed a conprehensive view of intra- and inter-chromosomal contacts between specific genomic sites. The locus control region of the {beta}-globin genes ({beta}-LCR) is a super-enhancer that is capable of activating all of the {beta}-like globin genes within the locus in cis through physical interaction by forming DNA loops. CTCF helps to mediate loop formation between LCR-HS5 and 3HS1 in the human {beta}-globin locus, in this way thought to contribute to the formation of a \"chromatin hub\". The {beta}-globin locus is also in close physical proximity to other erythrocyte-specific genes located long distances away on the same chromosome. In this case, erythrocyte-specific genes gather together at a shared \"transcription factory\" for co-transcription. Theoretically, enhancers could also activate target gene promoters on different chromosomes in trans, a phenomenon originally described as transvection in Drosophilla. Although close physical proximity has been reported for the {beta}-LCR and the {beta}-like globin genes when integrated at the mouse homologous loci in trans, their structural and functional interactions were found to be rare, possibly because a lack of suitable regulatory elements that might facilitate trans interactions. Therefore, we re-evaluated presumptive transvection-like enhancer-promoter communication by introducing CTCF binding sites and erythrocyte-specific transcription units into both LCR-enhancer and {beta}-promoter alleles, each inserted into the mouse ROSA26 locus on separate chromosomes. Following cross-mating of mice to place the two mutant loci at the identical chromosomal position and into active chromation in trans, their transcriptional output was evaluated. The results demonstrate that there was no significant functional association between the LCR and the {beta}-globin gene in trans even in this idealized experimental context.

genomics↗