Dierks, C. | bioRxiv Science

bioRxiv2025

In Ovo Sexing and Genotyping using PCR techniques: A Contribution to the 3R Principles in Chicken Breeding

Early sex determination and genotyping of chicken embryos is crucial for ethical and resource-efficient animal research, addressing concerns about surplus animals. We developed a reproducible workflow using whole genome amplification combined with Kompetitive Allele Specific PCR (KASP) and standard endpoint PCR to perform in ovo sexing and genotyping from embryonic day four (ED4/96h) onwards. The overall efficiency improved with embryonic age. Both standard PCR and KASP provided high success for sexing and genotyping (70-100% of samples yielding a result) and accuracy (92-100%) across multiple chicken lines, including a genetically modified line. Optimal reliability and hatchability were achieved when sampling at ED7. These PCR-based techniques enable precise early identification of sex and genotype, allowing selective removal of unwanted embryonated eggs before the assumed onset of nociception. This approach supports more humane and efficient practices in chicken breeding and research, contributing to the replacement, reduction and refinement (3R) principles in animal experimentation.

developmental biology↗

bioRxiv2025

A platform of robust patient-derived leukemia models covering subgroups for which no cell lines exist

Preclinical cancer research requires robust model systems, especially for poor prognosis entities like acute myeloid leukemia (AML), a highly aggressive blood cancer. Here, primary tumor cells from 137 AML patients of all age groups were transplanted into immune compromised mice to generate patient-derived xenografts (PDX). From these, 23 models enable robust, virtually endless serial re-transplantation and are amenable to lentiviral genetic engineering (*PDX AML models). These models primarily originate from patients with highly aggressive, relapsed disease. Comprehensive genomic, transcriptomic, and epigenomic analyses confirmed that they replicate primary AML biology more faithfully than conventional cell lines. Notably, *PDX AML models include AML subgroups that are underrepresented or absent in existing model systems, such as cytogenetically normal or IDH1/2-mutant AML. They withstand freeze-thaw cycles, making them suitable for broad distribution and reproducibility across research institutions. Luciferase-based in vivo imaging enables real-time monitoring of tumor progression and treatment responses in preclinical trials. Surprisingly, long-term treatment, including repeated cytarabine therapy over a period of one year, showed a gradual reduction in leukemia cell proliferation, which decreased continuously after each treatment block. Collectively, our *PDX models represent a robust, versatile, and relevant platform that holds great promise to accelerate translational research for the benefit of cancer patients. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/677299v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1ead31eorg.highwire.dtl.DTLVardef@1ca702corg.highwire.dtl.DTLVardef@18a7137org.highwire.dtl.DTLVardef@edbe6_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIWe present new robust AML PDX models covering subgroups for which no cell lines exist for use in various ex vivo and in vivo applications. C_LIO_LI*PDX models enable serial transplantation, genetic engineering and better representation of primary AML biology than cell lines. C_LIO_LIOne-year in vivo trials mimicking clinical chemotherapy showed surprising gradual decline in leukemia growth after each treatment block. C_LI

cancer biology↗