Production of sex-linked dwarf chickens via primordial germ cells modified by CRISPR-Cas9 system

報告時間：2024-04-12
報告地點：Room 407
指導老師：Pin Chi Tang
學生： Jia Jou Chou
摘要

Primordial germ cells (PGCs), the precursor of spermatogonia and oogonia, serve as the carriers of genetic materials. Chicken PGCs (cPGCs) exhibit a unique migration pattern. They are derived from the epiblast, and move to the hypoblast and enter the circulatory system at the HH13-15 stage. Eventually, cPGCs migrate and settle into the germinal ridge at the HH28-30 stage. After that, cPGCs gradually differentiate into the germ cells. During this transition period, cPGCs undergo significant proliferation. Due to the ability to carry genetic materials and specific migration, cPGCs have become a powerful tool to produce gene-edited animals recently. Previous studies have found that mutations in the growth hormone receptor (GHR) gene in chickens lead to the dwarf phenotype, characterized by decreased body weight and shortened shank. Also, as GHR gene is located on the Z chromosome, it is sex-linked dwarf (SLD). SLD chickens are beneficial to breeding management because they have lower feed and space consumption and have no difference in their egg production and offspring performance compared to their normal siblings. Therefore, this study aimed to investigate the differences in SLD traits and gene expression at the early embryonic stages, in addition to establishment of cPGC lines from Taiwan Country Chicken (TCC) B line and generation of SLD B line chicken by editing GHR gene using CRISPR/Cas9 system. Results showed that the growth performance of TCC dwarf L2 (dL2) line was inferior at embryonic Days 8,12, and the day of hatching, compared with those of TCC L2 line. However, there was no significant difference in the expression of insulin-like growth factor 1 (Igf-1), a downstream gene of GHR, in either muscle or liver tissues between the dwarf L2 and L2 strains. The present result is contrary to previous study in adult chicken, where Igf-1 expression was lower in dwarf chickens. It requires further investigation. Gonads isolated from 5.5-day-old chicken embryos were digested with trypsin and cultured on mouse fetal fibroblast (MEF) feeder layers. During the culture process, gonadal stroma cells gradually underwent apoptosis while cPGCs continued to proliferate. After several passages, when the cell number of cPGCs exceeded 106, they were cryopreserved. Thawed cPGCs injected into the dorsal artery of 2.5-day-old chicken embryos were found near the germinal ridge three days later. Subsequently, the GHR CRISPR-Cas9 construct will be transfected into cultured cPGCs, which will be then transplanted into the dorsal region of E2.5 chicken embryos to produce gonad chimeras. Through subsequent generations, GHR-deficient offspring will be bred by self-crossing to establish a population of dwarf B strain chickens.
 
Keywords: Sex-linked dwarf, Primordial germ cell, Growth hormone receptor, Taiwan Country Chicken
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