Localization of surfactant-associated proteins SP-A and SP-B mRNA in rabbit fetal lung tissue by in situ hybridization.
Localization of surfactant-associated proteins SP-A and SP-B mRNA in rabbit fetal lung tissue by in situ hybridization.
复制标题
通过原位杂交对兔胎肺组织中表面活性剂相关蛋白 SP-A 和 SP-B mRNA 进行定位。
DOI：
10.1165/ajrcmb/7.3.335
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发表时间：
1992
影响因子：
6.4
通讯作者：
Snyder,JM
中科院分区：
文献类型：
--
作者：
Wohlford-Lenane,CL;Snyder,JM
动物妊娠新西兰兔从Knapp Creek Rabbitry(Amana，IA)获得，并在爱荷华大学批准的动物护理设施中饲养。用于进行这项研究的实验方案得到了爱荷华大学动物研究审查委员会的批准。用戊巴比妥钠溶液(10毫升，50毫克/毫升)经耳静脉快速注射处死妊娠兔。取下胎儿，斩首，自由解剖胎儿肺，并将每个肺的右上叶迅速冷冻在液氮中。分别于妊娠第19、21、24、26、28、31天(足月=31天)处死妊娠兔。此外，还取第3天新生动物和成年兔的肺组织。所有组织都保存在-70℃，直到用于分析。胎肺组织取自至少三个不同孕龄的孕兔，以及至少三个不同的新生和成年兔。1.9kb的兔SP-A cDNA探针是C.R.Mendelson博士和V.Boggaram(达拉斯，德克萨斯州)(11)赠送的一种礼物。本实验室将兔SP-AcDNA探针亚克隆到pGEM载体(24)中。1.7kb的兔SP-B基因是加拿大安大略省伦敦市F.Possmayer博士赠送的礼物(14)。将含有SP-A基因的载体分别用PvuⅡ和Hind III双酶切，合成正义和反义SP-A cRNA转录本。用Cla II和Not I线性化含有SP-B基因的蓝本载体，分别合成正、反义SP-B cRNA转录本。用[~3H]-CTP(4.82×107dpm/ug)和PH]-UTP(5.87×107dpm/ug)合成RNA转录本(均为新英格兰核，波士顿，马萨诸塞州)。使用RNA转录试剂盒(Gemini Ribobe System II；Promega，Madison，WI)、1~g消化的质粒DNA和适当的RNA聚合酶进行标记反应。放射性标记的cRNA探针随后被水解为大约300个碱基的片段，正如Cox和他的同事所描述的(25)。
Materials and MethodsAnimals Pregnant New Zealand rabbits were obtained from Knapp Creek Rabbitry (Amana, IA) and were maintained in an approved animal care facility at the University ofIowa. The experimental protocols used to perform this study were approved by the Animal Research Review Committee at the University of Iowa. Pregnant rabbits were killed by the rapid injection of a solution of pentobarbitol (10 ml, 50 mg/ml) into an ear vein. The fetuses were removed, decapitated, the fetal lungs dissected free, and the right upper lobe of each lung was quickly frozen in liquid nitrogen. Pregnant rabbits were killed on days 19, 21, 24, 26, 28, and 31 of gestation (term= 31 days). In addition, lung tissue was also obtained from day 3 neonatal animals and from adult rabbits. All tissues were stored at-70 C until used for analysis. Fetal lung tissues were obtained from at least three different pregnant does at each gestational age and from at least three different neonatal and adult rabbits. cDNA Probes The 1.9-kb rabbit SP-A cDNA probe was a kind gift from Drs. C. R. Mendelson and V. Boggaram (Dallas, TX)(11). The rabbit SP-AcDNA probe was subcloned in our laboratory into a pGEM vector (24). The 1.7-kb rabbit SP-B cDNA, in a Bluescript vector, was a kind gift from Dr. F. Possmayer (London, Ontario, Canada)(14). The vector that contained the SP-A cDNA was digested with Pvu II and Hind III for the synthesis of the sense and antisense SP-A cRNA transcripts, respectively. The Bluescript vector that contained the SP-B cDNA was linearized with Cla II and Not I and utilized to synthesize the sense and antisense SP-B cRNA transcripts, respectively. RNA transcripts were synthesized using [3H]-CTP (4.82 X 107 dpm/ug) and PH]-UTP (5.87 x 107 dpm/ug)(both New England Nuclear, Boston, MA). The labeling reaction was performed using an RNA transcription kit (Gemini Riboprobe System II; Promega, Madison, WI), 1~ g of the digested plasmid DNA, and the appropriate RNA polymerase. The radiolabeled cRNA probes were subsequently hydrolyzed to approximately 300-bp fragments as described by Cox and colleagues (25).