GoKawiilAnimal experiments
All animal work in this study was approved by the Animal Care Committee of The Centre for Phenogenomics (TCP) (AUP 25-0100H) and by the Institutional Animal Care and Use Committee of the Center for Comparative Medicine (CCM) at Baylor College of Medicine (AN-9134 and AN-9136). All mice housed at TCP were maintained under standard housing conditions with a 12-h light–12-h dark cycle (lights on at 07:00; lights off at 19:00), an ambient temperature of 21–23 °C and relative humidity of 40–60%. All mice housed at CCM were maintained under standard housing conditions with a 12-h light–12-h dark cycle (lights on at 06:00; lights off at 18:00), an ambient temperature of 20–22 °C and relative humidity of 30–70%. All mice had ad libitum access to food and water. Timed breeding of C57BL/6J mice was set up, and whole litters were collected from eight independent embryonic time points: E9, E10, E11, E12, E13, E14, E16 and E18. Sexes for all embryos were determined from tail clippings using PCR protocols. Tissues from two female and two male littermates were selected at each time point to undergo dissociation.
FCG experiments
The FCG mice were originally provided by J. Danska, and additional mice were purchased from The Jackson Laboratory (strain 039108). The strain was maintained on a C57BL/6J background. XY mice with testes (XY−-Sry) were bred with wild-type C57BL/6J females to generate the four types of offspring. Caudal cerebellum was collected from E16 and E18 embryos. Genotypes for all embryos were determined from tail clippings using PCR protocols. Cerebella from one XX embryo with ovaries (XX), one XX embryo with testes (XX-Sry), one XY embryo with ovaries (XY−) and one XY embryo with testes (XY−-Sry) were collected at each time point for tissue dissociation.
DNA extraction
DNA was extracted from mouse tissue using the QIAGEN DNeasy Blood and Tissue Kit (QIAGEN, 69506) according to the manufacturer’s instructions.
PCR reactions
Genomic DNA was amplified with the following primer pairs: SX_F (5′-GATGATTTGAGTGGAAATGTGAGGTA-3′), SX_R (5′-CTTATGTTTATAGGCATGCACCATGTA-3′), SRY_F (5′-AGCCCTACAGCCACATGATA-3′) and SRY_R (5′-GTCTTGCCTGTATGTGATGG-3′). X and Y chromosomes were distinguished by PCR as previously described53. In brief, SX_F and SX_R amplified the pseudoautosomal Sly and Xlr genes on the Y and X chromosome, respectively. The presence or absence of the Sry gene in FCG offspring was determined using the SRY_F and SRY_R primers. PCR reactions were performed in a final volume of 20 µl with 1.5 mM MgCl 2 , 200 µM dNTPs, 0.5 µM primers, 0.4 µl of Phire Hot Start II DNA Polymerase (Thermo Fisher Scientific, F-122L) and the following PCR parameters: initial denaturation at 98 °C for 30 s, 30 cycles with denaturation at 98 °C for 5 s, annealing at 60 °C for 5 s and extension at 72 °C for 5 s, followed by final extension at 72 °C for 1 min. PCR products were analysed on a 1.5% agarose gel with a 100-bp DNA ladder (FroggaBio, DM001-R500) and visualized with ethidium bromide.
Patient-derived PFA-EPN tissues
All PFA-EPN tissues used in this study were obtained with properly informed consent of patients. All experimental procedures were performed in accordance with the Research Ethics Boards (REB 1000055059) at The Hospital for Sick Children.
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