Irs1^S57X Heterozygous Mutant Mice Display Normal Hematopoiesis and Phenotypic Features, While Homozygous Knockout Exhibit High Fetal or Postnatal Lethality

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-Irs1^smla mouse carries a spontaneous nonsense mutation in serine 57 (Irs1^S57X), 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 Irs1^smla 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 Irs1^S57X 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-Irs1^smla/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 this genotype in the statistical analysis. The single Irs1 knockout mouse that survived to weaning was euthanized at 9 weeks of age for analysis. The Irs1-deficient mouse was male and characterized by low body weight (15.64 grams); 6.8x10⁹/LWBC; 9.79x10¹²/L RBC, 16.4 g/dl HGB; 48.5% HCT and 311x10⁹/Lplatelets. Wild type animals and heterozygotes were followed up for four months from 8 weeks of age, being weighed and submitted to peripheral blood analysis monthly. Among wild type animals (n= 7 females and 4 males) and heterozygotes (n= 7 females and 12 males) there were no significant differences in body weight and hematological parameters of peripheral blood, at all age ranges evaluated (all p>0.05). Irs1^S57X mutation, in homozygosis, promotes fetal or postnatal lethality. Progeny genotyping demonstrated that homozygous fetuses developed until gestational day 18.5. Full-term deliveries (n=2 females) resulted in 17 pups, including 11 born alive and 6 miscarried/dead immediately after birth; the stillborn were exclusively homozygous.

Conclusions: Irs1^S57X mutation, in heterozygous state, does not result in phenotypic alterations in weight and hematological parameters. In this study, spontaneous truncated homozygosis mutation Irs1^S57X, from a pure C57BL/6 genetic background, resulted in high fetal or postnatal lethality. High mortality was also described in a knockout model for SerpinB2, which spontaneously acquired the Irs1 S57X mutation, resulting in the total loss of Irs1 protein expression (WESTRICK et al., 2010). Irs1-deficient models generated by targeted disruption do not exhibit significative reduction in survival (ARAKI et al., 1994; TAMEMOTO et al., 1994). These data suggest that the elevated mortality observed in this study may not directly be related to Irs1 deficiency. Based on these findings, further hematologic studies of B6.129S2-Irs1^smla/J mice should be focused on fetal hematopoiesis.