Rescue of Hemostasis-deficiency States

因子X(FX)缺陷的胚胎会出现部分胚胎死亡，大约30%的胚胎会在中期停滞。剩下的动物存活到足月，但主要死于腹部或颅内出血。我们已经通过将FX+/+rosa26胎儿的肝细胞移植到Fx+/-杂合子杂交衍生的中期胚胎中来拯救Fx缺陷小鼠。FX-/-胚胎以预期的频率出生，50%的FX-/-新生儿存活超过4个月。注射生理盐水存活到足月的FX-/-胚胎在围产期死亡，类似于未经处理的FX-/-小鼠。在获救的16周FX-/-基因小鼠中，血浆FX水平大约是年龄匹配的野生型小鼠的2-5% 级别。来自供体ROSA26胎肝细胞的β-半乳糖苷酶染色细胞在大约45%的成年小鼠的肝脏中被检测到。观察表明，这种抢救方法可以用于治疗在子宫中发现的潜在血友病患者。这项提案的目的是更好地定义和修改这一程序，以开发：1)治疗肝脏表达蛋白缺陷的治疗策略；2)有条件地挽救肝细胞癌的研究方案。 关键止血因素。后一个目标包括确定供体细胞群体的特征，以确定具有肝定植特性的细胞亚群。还建议开发具有定植活性的培养细胞系，从而消除对原代细胞的需要。此外，将对供体细胞进行基因改造，以提高其对这一策略的有效性。这将涉及增加目标因子的表达水平，或增加供体细胞系的肝脏定植潜力。将负选择标记引入供体细胞群体将使供体细胞得以根除。 人口在选定的时间。这使得这种方法可以作为一种有条件的救援策略来研究成年动物特定基因缺失的生理后果，而同样的缺失通常会导致胚胎或围产期死亡。特别是，这一策略将被用于研究体内FX缺乏对老年小鼠的病理生理学后果，这是目前无法完成的壮举。

Factor X (FX)-deficient embryos suffer partial embryonic lethality with approximately 30% of the embryos arresting at midgestation. The remaining animals survive to term, but die perinatally mainly from abdominal or intracranial hemorrhage. We have rescued FX-deficient mice by transplanting liver cells from FX+/+ Rosa26 fetuses into midgestation embryos-derived from FX+/- heterozygous crosses. FX-/- embryos were born at the expected frequency and 50% of the FX-/- neonates survived longer than 4 months. FX-/- embryos receiving saline injections that survived to term died perinataily, similar to untreated FX-/- mice. The plasma levels of FX in the rescued 16-week genotypically FX-/- mice were approximately 2-5% of age-matched wild-type levels. Beta-galactosidase-staining cells, derived from the donor Rosa26 fetal liver cells, were detected in approximately 45% of the livers of adult mice. The observations suggest that this rescue approach can be used to treat potential hemophiliacs detected in utero. The aims of this proposal are to better define and modify the procedure to develop: 1) a therapeutic strategy to treat deficiencies of liver-expressed proteins; 2) a research protocol for conditional rescue of critical hemostatic factors. This latter aim involves characterizing the donor cell population to identify the cell subpopulations possessing liver-colonizing properties. It is also proposed to develop cultured cell lines with colonizing activity, thus eliminating the need for primary cells. Furthermore, the donor cells will be modified genetically to increase their efficacy for this strategy. This will involve increasing the levels of expression of the factor of interest, or increasing the liver-colonizing potential of the donor cell lines. The introduction of negative selection markers into the donor cell population would enable the eradication of the donor cell population at a chosen time. This permits the approach to be used as a conditional rescue strategy to study the physiological consequences of the deficiency of particular genes in adult animals, when the same deficiency would normally result in embryonic or perinatal lethality. In particular, this strategy will be used to study the pathophysiological consequences of FX deficiency in vivo in older mice, a feat that cannot be accomplished at present.