95 Obtaining birds with chimeric gonads using in vitro lentiviral transduction of primordial germ cells
K. A. Glumakova A , O. N. Mityaeva A , E. N. Antonova A , O. V. Glazova A , A. S. Komarchev B and P. Y. Volchkov AA Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia;
B Russian State Agrarian University, Moscow Timiryazev Agricultural Academy, Moscow, Russia
Reproduction, Fertility and Development 32(2) 173-173 https://doi.org/10.1071/RDv32n2Ab95
Published: 2 December 2019
Abstract
Avian primordial germ cells (PGCs) have unique migration capacity towards the gonads via the bloodstream. Therefore, PGCs in vitro genome modification is a dominant approach for poultry genetic modification. The aim of this study was to improve cultivation conditions of PGCs in terms of proliferation activity and further analyse their migration abilities. We isolated PGCs from whole blood cells collected from the embryonic dorsal aorta (Hamburger-Hamilton (HH) stage 14-17) and cultured them without feeder cells in customized avian knockout Dulbecco's modified Eagle's medium basal medium supplemented with fibroblast growth factor 2, Activin A and B-27 supplement containing insulin. We analysed the number of cells during the month of culture. Proliferation of PGCs increased in the first 7 d of culture when both BMP4 and Activin A growth factors were added to the medium (four out of seven samples). Kinetic of stemness and PGC-marker genes (Nanog, PouV, DAZL) were similar to expression in mature gonadal PGCs (gPGCs). Expression levels of marker genes were significantly greater in the freshly isolated PGCs compared to the gPGCs and decreased during cultivation. In order to confirm the migration activity of the cultivated PGCs, cells were labelled with lentivirus (ZsGreen) and injected into the embryo blood stream (HH 14-17). Gonads from the recipient embryos were retrieved (HH 20-26) and analysed. Due to the low level of fluorescence in mature cells, the presence of transgenic PGC in the gonads was detected by ZsGreen-specific quantitative PCR. We confirmed that the cultured cells maintained migration activity, and efficient engraftment was detected in 75% of embryos.
