Irs1S57X Heterozygous Mutant Mice Display Normal Hematopoiesis and Phenotypic Features, While Homozygous Knockout Exhibit High Fetal or Postnatal Lethality

• Published in: Blood 2020-11, Vol.136 (Supplement 1), p.33-34
• Main Authors: Fenerich, Bruna Alves, Fernandes, Jaqueline Cristina, Silva, Cleide Lúcia Araújo, Coelho-Silva, Juan L, Silva, Antonio Bruno Alves, Fonseca, Natasha Peixoto, Machado-Neto, João Agostinho, Traina, Fabiola
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2020-139422

Introduction: Pharmacological inhibition of insulin receptor substrate 1 (IRS1) protein has been demonstrated to promote antineoplastic effects in hematological disorders, including myeloproliferative neoplasms, chronic myeloid leukemia and acute lymphoblastic leukemia. However, the role of IRS1 in normal hematopoiesis has not yet been elucidated. In this scenario, using a murine Irs1 knockout model represents an interesting tool to evaluate the role of this gene in normal hematopoiesis. B6.129S2-Irs1smla mouse carries a spontaneous nonsense mutation in serine 57 (Irs1S57X), which in homozygosis produces a knockout animal for Irs1, characterized by a small size. Throughout the development of the study, we noticed that the successive mating between Irs1smla heterozygous animals did not result in homozygous mice among the offspring. In view of these findings, the present study aimed to compare the hematological parameters of wild type and heterozygous mice for the Irs1S57X mutation and to describe the fetal lethality of homozygous mice. Methods: Protocols for mouse experiments were approved by the Animal Ethics Committee of the Institution. B6.129S2-Irs1smla/J mice (Jackson Laboratory, stock number: 007240) from a pure C57BL/6 background were mated to produce homozygous animals. For peripheral blood analysis, mice were evaluated monthly at different age ranges (8-10; 12-14; 16-18; 20-22 weeks of age). Samples were collected in heparin and submitted to automated blood cell count. The hematological parameters evaluated included white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT) and platelets. For fetal lethality evaluation, mating was performed between heterozygous mice. Females were followed up daily to check for the presence of a vaginal plug. The time that the plug was identified was considered as 0.5 days post coitus (dpc) or D+0.5. After the plug was found, the females were accommodated separately until the date of euthanasia. Euthanasia of the females was performed on gestational days D+9.5 (n=1), D+12.5 (n=1), D+15.5 (n=2) and D+18.5 (n=2). The fetuses were carefully separated from the maternal material to avoid contamination. Genotyping was performed by Sanger sequencing. Results: After 28 intercrossing events, a total of 101 heterozygotes, 46 wild type mice and 1 homozygous mouse were obtained, with an average of 4.9 (± 1.6) born alive per female. The absence of homozygous knockout adult mice precluded the inclusion of