Novel methods to avoid difficulties from juvenile lethality or growth retardation in knockout mice and to study circulatory, respiratory, and autonomic functions in them

为了避免基因敲除小鼠幼年致死或生长迟缓带来的困难，并研究它们的循环、呼吸和自主神经功能，人们开发了五种方法：从新生小鼠制作延髓-脊髓准备，通过气管切开拯救新生幼鼠，测量血压和呼吸力学的特殊小装置，构建人工肛门，以及遗传拯救。这些方法被应用于ET-1基因敲除小鼠、ET-A受体(ETAR)基因敲除小鼠、ET-B受体(ETBR)基因敲除小鼠和胰岛素受体底物-1(IRS-1)基因敲除小鼠。ET-1基因敲除的小鼠和气管切开的ET-1基因敲除和ETAR基因敲除的新生小鼠的延髓-脊髓标本显示呼吸反射对高碳酸血症的严重减弱。因此，内源性ET-1-Etar系统在生理上参与了中枢化疗敏感性。在ETBR为正常小鼠的1/8的遗传突变小鼠中，动脉血压(AP)升高，交感神经活动和呼吸正常。这一结果与选择性阻断ETBR对正常小鼠的升压反应一致。因此，ETBR通过外周机制生理性地降低AP的基础水平。IRS-1基因敲除小鼠表现出生长迟缓和AP升高，因此IRS-1也是维持正常AP所必需的。综上所述，IRS-1基因敲除小鼠在哺乳动物综合生理功能的研究中是一种有价值的研究资源。

Five methods were developed to avoid difficulties from juvenile lethality or growth retardation in knockout mice and to study circulatory, respiratory, and autonomic functions in them : to make medulla-spinal cord preparation from newborn mice, a way to rescue newborn pups with tracheotomy, special small devices to measure blood pressure and respiratory mechanics, construction of artificial anus, and genetic rescue. These methods were applied to endothelin (ET) -1 knockout mouse, ET-A receptor (ETAR) knockout mouse, ET-B receptor (ETBR) knockout mouse, and insulin receptor substrate-1 (IRS-1) knockout mouse. Medulla-spinal cord preparation from ET-1 knockout mice and tracheotomized newborn mice of ET-1 knockout and ETAR knockout showed severe attenuation of respiratory reflex to hypercapnia. Endogenous ET-1-ETAR system thus physiologically participates in the central chemosensitivity. In genetically established mutant mice in which ETBR was 1/8 of normal mice, arterial blood pressure (AP) was elevated with normal sympathetic activity and respiration. The result is consistent with a pressor response to selective blockade of ETBR in normal mice. Thus, ETBR contribute physiologically to diminish basal level of AP through peripheral mechanism. IRS-1 knockout mice showed growth retardation and elevation of AP.Thus IRS-1 is also essential to maintain normal AP.In conclusion, knockout mice are a valuable research resource especially in the study of integrative physiological functions of mammals.