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A method of studying the related gene function of spinal cord neural stem cells based onin ovo electroporation for chicken embryos
YU Ya-nan LIU Yan-li YANG Ci-qing ZHANG Bi-chao XU Zhen-ping LIN Jun-tang*
Acta Anatomica Sinica ›› 2016 ›› Issue (3) : 415-420.
A method of studying the related gene function of spinal cord neural stem cells based onin ovo electroporation for chicken embryos
Objective To develop a method of studying the related gene function of the chick spinal cord neural stem cells (NSCs) based onin ovo electroporation for chicken embryos. Methods The surface markers of the spinal cord NSCs were tested by RT-PCR at the chick different developmental stages. Subsequently, the pCAGGS-GFP plasmid was transfected into the chick spinal cord using in ovoelectroporation when the fertilized eggs were cultured for E2.5-E3; GFP-positive chick embryos were collected at E6, five samples in each group at least. The specimens were either transversely sectioned or undergone “open-book” processing. The commissural axons were observed under a fluorescent microscope. Finally, the spinal cords were isolated under the stereomicroscope, and the NSCs were cultured in 37℃ and 5%CO2 condition after trypsin digest. Results RT-PCR results showed that the NSCs surface markers were expressed in chick spinal cords; the subsequent results of transverse sections and open-book processing collectively showed that GFP labeling commissural axons crossed the midline region through the floor plate to the contralateral side of the spinal cord. The GFP labeling cells isolated from spinal cords exhibited typical morphological characteristics of NSCs, and generated neuritis when cultured in vitro. Conclusion A new method is successfully established, which could be used to study the gene function of the chick spinal cord NSCs based onin ovo electroporation.
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