Identification of transgene integration site and anatomical properties of fluorescence intensity in a EGFP transgenic chicken line

Abstract

Transgenic birds are commonly used for time‐lapse imaging and fate mapping studies in developmental biology. When researchers use transgenic birds expressing fluorescent protein, they need to understand the integration site of the transgene in the genome and the intensity of fluorescence in the tissues of interest. In this study, we determined the integration site of the transgene and fluorescence property of developing organs in our transgenic chicken line generated by lentivirus infection. The transgene was localized between exons 3 and 4 of MED27. Some homozygotes and heterozygotes appeared to be lethal at early embryonic stages. We performed histological analysis of EGFP expression in transgenic embryos at St. 14, 17, and 24 by immunohistochemistry with anti‐GFP antibody on paraffin sections. Next, we cut cryosections and quantified direct EGFP intensity from the transgene in each tissue without performing immunohistochemistry. These results revealed that EGFP intensity in each tissue was unique in developing embryos and changed according to developmental stages. Finally, we demonstrated that EGFP‐expressing cells in a micromass culture with co‐culturing wild‐type cells were clearly distinguishable via live cell imaging. These results provide essential information on the potential of our transgenic line and indicate that these transgenic chicken lines are useful for research associated with developmental biology.

Mutation Screening of MED27 in a Large Dystonia Cohort

“…The symptoms of the patients carrying heterozygous variant in MED27 detected in the current study were much milder than the individuals carrying biallelic variants of MED27 as the original study reported [4]. A study found that both homozygous and heterozygous mutation of MED27 can be lethal at early embryonic stage in chicken [17]. Therefore, the identified heterozygous mutations might play a role in causing milder symptoms compared to biallelic mutations in our patients here due to a potential haploinsufficiency of MED27.…”

MED27 Variants Cause Developmental Delay, Dystonia, and Cerebellar Hypoplasia

“…In flies, Med27 knockout caused lethality at the pupal stage 17,18 . Similarly, chickens carrying homozygous MED27 insertional truncating mutations were born at less than expected Mendelian ratios, suggesting partial embryonic lethality in homozygotes 19 . Although the specific biological function of MED27 remains unknown, it clearly plays an essential role in early embryonic and neuronal development.…”

Transcription Pause and Escape in Neurodevelopmental Disorders

“…Indeed, in zebrafish, Med27 loss of function disrupts dopaminergic amacrine cells and serotonergic neurons resulting in size reduction of head, eye, jaw, and eventually leading to lethality 6 days post fertilization ( Durr et al, 2006 ). In fly and chicken, disruption of Med27 also leads to embryonic lethality ( Gokcezade et al, 2014 ; Li-Kroeger et al, 2018 ; Tsujino et al, 2019 ). Together these results clearly indicate an important role for Med27 in embryonic and neuronal development.…”