疾病动物模型,Duox2基因突变 z-bio 2021-03-11 312

　　[Modeling mechanism] In the synthesis of thyroxine, the iodination reaction of thyroglobulin requires the catalysis of hydrogen peroxide and thyroid peroxidase. Dual oxidases (DUOX) provide hydrogen peroxide in the process. Mutations in the DUOX2 gene can cause hypothyroidism.

　　Spontaneous mutations were found in the B6.129-Tnfrsfla tmlMak/J mouse congener. Genetic analysis revealed a mutation in exon 16 of the Duox2 gene on the second pair of chromosomes.

　　[Method of Modeling] After the discovery of Tnfrsfla tmlMak mutant mice, the thyd mutant gene was introduced into the C57BL/6J background and named B6(129)-Duox2thyd/J. Homozygous mutant mice cannot be bred by themselves. They need to be bred with male C57BL/6J through ovarian transplantation, and the mutant gene is introduced into C57BL/6J mice to preserve the species as heterozygotes. The heterozygotes mate with each other to produce homozygous mutant mice for experiments.

　　[Model Features] Homozygous thyd mutant mice are smaller than other mice in the same litter. Adult 8-week-old mutant mice have shorter long bones, low bone density, incomplete ossification, less subcutaneous fat, and low serum IGF-1.

　　mutant mice have abnormal pituitary gland and thyroid gland. The goiter is enlarged with only a few normal follicles; the neurohypophysis and middle lobe are normal, but the anterior lobe is underdeveloped and contains a lot of abnormal cells. Serum T4 is 10 times lower than normal control. TSH is 100-1000 times higher than normal control.

　　mutant mice have impaired hearing, the hearing stimulation response is 50-60dB higher than the normal control, and the cochlear development is retarded.

　　[Model evaluation and application] Spontaneous hypothyroidism mouse model is mainly used for the study of hereditary congenital hypothyroidism due to the mutation of Duox2 gene which affects the biosynthesis of thyroxine and causes hypothyroidism.

　　This model can be applied to research on the biosynthesis of thyroxine and the pathogenesis of hypothyroidism. Because the DUOX2 gene mutation not only affects the synthesis of thyroxine, but also affects the development of other organs, such as the development of the pituitary gland and the ossification of long bones, it is not completely consistent with the clinical manifestations of hypothyroidism. This model should be used to analyze the experimental results. 3. Genetically modified mouse model of hypothyroidism

　　1. TTF-1 gene knockout hypothyroidism mouse model

　　[Modeling mechanism] Thyroid-specific transcription factor 1 (TTF-1), also known as thyroid-specific enhancer-binding protein, is a member of the transcription factor family. Contains homeobox domains. TTF-1 is extremely important in the formation of embryonic organs. Knockout of TTF-1 gene will result in stillbirth, lack of thyroid and pituitary glands, ventral and base defects of lung and forebrain, and hypoplasia of globus pallidus.

　　TTF-1 gene expresses three proteins, thyroid peroxidase, thyroglobulin and thyroid-stimulating hormone receptor. These three proteins are essential for the biosynthesis of thyroxine. , TTF-1 gene knockout leads to congenital hypothyroidism.

　　[Modeling method] A total of 5.9 kb containing the entire coding region of the TTF-1 gene and the 3'non-coding region sequence is used to construct a targeting vector. The insertion of the neo gene into the second exon of the TTF-1 gene is being screened, resulting in The first helix of the homologous domain sequence is destroyed; the tk expression cassette is used for negative selection of non-homologous recombination. The selected integrated ES cell clones were microinjected into the blastocysts of C57BL/6 mice to produce Nine-zygote mice. The 5/9 male chimera mice passed the ES cell genome to their offspring.

　　[Model Features] Heterozygous mice develop normally. Homozygous mice die at birth, have no thyroid, but have normal parathyroid glands, lack lung parenchyma, but have basic bronchial trees, pleural epithelial abnormalities, and extensive defects in the brain of homozygous mice, especially in the forebrain abdomen. The entire pituitary (including the anterior, middle and posterior lobe) is lacking. In situ hybridization showed that TTF-1 gene was expressed in specific regions of normal thyroid, lung bronchial epithelium and forebrain during early embryogenesis.

　　【Model evaluation and application】This model can be used for the study of fetal development in congenital hypothyroidism. Because TTF-1 plays a role in the formation of multiple organs of the embryo, knockout of TTF-1 not only affects the synthesis of thyroxine, but also affects the formation of other organs (such as the brain, pituitary gland, lungs, etc.), which is not completely consistent with congenital nails. Attention should be paid to the clinical manifestations of reduced symptoms when using this model to analyze the experimental results.

　　2. Pax8 gene knockout hypothyroidism mouse model

　　[Modeling mechanism] Some mouse genes contain highly conserved DNA sequences homologous to the paired box of Drosophila, called "Pax genes". Pax8 genes play an important role in organ formation. From the 10.5th day of pregnancy, Pax8 gene began to be expressed in the thyroid gland, and Pax8 gene knockout affected the formation of the thyroid gland.

　　[Modeling method] The second exon of Pax8 gene was replaced with pGKNeopA, and the tk gene was inserted into the 3'end of the targeting vector. [Model Features] Heterozygous Pax8 knockout mice are healthy and fertile. Heterozygotes cross each other to produce homozygotes. Homozygous children showed obvious developmental delay within 1 week after birth, and died after weaning. Histological analysis of fetal thyroid defects. Homozygous Pax8 knockout mice failed to detect the late gene expression of thyroglobulin and thyroid peroxidase encoded by thyroid follicular cells at 15.5 to 18.5 days of fetus. Other organs expressing Pax8, such as the spinal cord, midbrain/hindibrain, or kidney are normal.

　　Homozygous Pax8 knockout mice have smaller thyroid glands. No follicles. Immunohistochemistry showed that the thyroid gland of newborn mice was composed entirely of thyroid C cells. Serum thyroxine levels dropped significantly at 2 weeks of age, and supplementation of exogenous thyroxine can prolong survival to 6 months of age.

　　【Model evaluation and application】This model can be used for the study of fetal development in congenital hypothyroidism.

　　3. DHTP/B6 double gene knockout hypothyroidism mouse model

　　[Modeling mechanism] TTF-1 and Pax8 are important transcription factors for organogenesis and play an important role in thyroid differentiation. When these two transcription factors are defective at the same time, hypothyroidism can occur.

　　【Method of Modeling】Heterozygous TTF1 knockout mice and heterozygous Pax8 knockout mice are produced by mating, TTF1 and Pax8 double heterozygous DHTP mutant mice. The DHTP mice were backcrossed for 10 generations under the background of C57BL/6J. More than 99% of the genetic background came from C57BL/6J, resulting in heterozygous TTF1, heterozygous Pax8 and double heterozygous DHTP mutant mice. Double heterozygous DHTP mutant mice were used as models.

　　[Model Features] Compared with single gene knockout heterozygotes or wild-type DHTP/B6 mice, 3-month-old DHTP/B6 mice have significantly lower body weight and T4 levels. Histological examination of the thyroid gland was irregular and diffuse hyperplasia, and the follicular endothelium showed long columnar cells; immunochemistry showed that the expression of sodium iodide symporter in follicular cells was increased. Although the serum TSH concentration increased, the thyroid was not enlarged and was slightly smaller than the normal control group. About 30% of mice have unilateral thyroid absent, unilobular shape, slightly enlarged, normal position but only on one side of the trachea.

　　From the 15th to 16th day of pregnancy, the thyroid of the fetuses of DHTP/B6 mice was smaller than that of the wild-type control, and continued until the end of pregnancy. Approximately 30% of the fetal thyroid is unilobular, which is consistent with that observed in adult DHTP/B6 mice. Therefore, hypothyroidism and the unilateral absence observed in adult DHTP/B6 mice are congenital. On the 18th day of pregnancy, the thyroglobulin and iodothyronine deiodinase type I mRNA of DHTP/B6 mouse fetuses decreased significantly.

　　[Model Evaluation and Application] This model can be used for the research of primary hereditary congenital hypothyroidism, and can be used for thyroxine replacement therapy. Since DHTP/B6 mice are double heterozygous mutant mice, each experiment requires heterozygous TTF1, heterozygous Pax8 and wild-type as controls.

　　TTF-1 knockout mice and Pax8 knockout mice have normal heterozygotes. Homozygotes die at birth or at weaning. They are mainly used to study the impact of congenital hypothyroidism on fetuses.

　　TTF-1 and Pax8 double gene knockout double heterozygous mice (DHTP/B6) is a good hereditary congenital hypothyroidism model, which shows many clinical manifestations of human hereditary congenital hypothyroidism, and Thyroxine can be used for early replacement therapy.