Carmack C E, Camper S A, Mackle J J, Gerhard W U, Weigert M G
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111.
J Immunol. 1991 Sep 15;147(6):2024-33.
A rearranged murine V kappa 8/J kappa 5 L chain gene that codes for the L chain of most antibodies generated in the primary response of BALB/c mice to the antigenic site, Sb, of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8) has been cloned. Three transgenic lines were generated by microinjecting the gene. Lines Ga and L each contain a single copy of the transgene whereas line Gb contains three complete copies. Mice of the Ga lineage showed increased V kappa 8-specific mRNA levels only in spleen, but not in nonlymphoid organs and therefore displayed apparently normal lymphoid-specific regulation of the Ig transgene. B cell hybridomas generated from these mice were analyzed for rearrangements of endogenous V kappa genes. Greater than 90% of the C kappa alleles were retained in germ-line configuration in the Ga line, compared with only 0 to 18% in the L line. Thus, a wide variation in the frequency of endogenous rearrangements is seen among mice of different lineages using the same transgene construct. None of more than 150 hybridomas derived from LPS-stimulated splenic B cells of Ga mice exhibited HA-binding activity although they expressed the transgene and, in most cases, excluded endogenous V kappa rearrangements. In contrast, a large fraction of hybridomas isolated after primary immunization with PR8 were HA(Sb)-specific. This indicated that the transgene was functional but formed HA-specific antibodies with a more restricted set of H chains than previously hypothesized. The primary anti-HA response to immunization with PR8 was diminished in all lines compared with normal mice except for a slightly accelerated but transient burst of anti-HA antibody formation in two out of three lines (Ga and Gb). This early response in G lineage mice was largely specific for HA(Sb) and thus appeared to be composed of transgene-expressing antibodies. No differences in serum titers were observed in the secondary anti-HA responses to booster inoculation with PR8 between transgenic and normal mice.
一个重排的小鼠Vκ8/Jκ5轻链基因已被克隆，该基因编码BALB/c小鼠对甲型流感病毒A/PR/8/34（PR8）血凝素（HA）分子抗原位点Sb产生的大多数初次免疫应答抗体的轻链。通过显微注射该基因产生了三个转基因品系。Ga和L品系各含有一个转基因拷贝，而Gb品系含有三个完整拷贝。Ga品系的小鼠仅在脾脏中显示Vκ8特异性mRNA水平升高，而非淋巴器官中未升高，因此显示出Ig转基因明显正常的淋巴特异性调控。对这些小鼠产生的B细胞杂交瘤进行内源性Vκ基因重排分析。Ga品系中超过90%的Cκ等位基因保持在种系构型，而L品系中仅为0%至18%。因此，使用相同转基因构建体的不同品系小鼠之间，内源性重排频率存在很大差异。来自Ga小鼠LPS刺激的脾B细胞的150多个杂交瘤中，没有一个表现出HA结合活性，尽管它们表达了转基因，并且在大多数情况下排除了内源性Vκ重排。相反，用PR8初次免疫后分离的大部分杂交瘤是HA（Sb）特异性的。这表明转基因是有功能的，但形成的HA特异性抗体的重链组合比先前假设的更受限。与正常小鼠相比，所有品系对PR8免疫的初次抗HA应答均减弱，除了三个品系中的两个（Ga和Gb）有轻微加速但短暂的抗HA抗体形成爆发。G品系小鼠的这种早期应答在很大程度上对HA（Sb）具有特异性，因此似乎由表达转基因的抗体组成。转基因小鼠和正常小鼠对PR8加强接种的二次抗HA应答中，血清滴度没有差异。
