Rat FceR1a was amplified from complementary DNA (cDNA) prepared from total RBL-2H3 cell RNA using the oligonucleotide primers 5′-TAGTAGACGCGTGCCACCATGGATACTGGAGGATCTGCCCGGCTG-3′ and 5′-CTACTAGGTACCGCCACTCCCACCTTTTTTTTTGCCTTTTCCAGTC-3′ and subcloned into the lentiviral pHR vector upstream of a SNAP-tag–encoding sequence. The resultant construct contained a flexible Gly-Ser-Gly sequence between FceR1a and SNAP-tag. Syk-citrine was also generated in pHR by amplification of rat Syk from RBL-2H3 cDNA using the primers 5′-TAGTAGACGCGTGCCACCATGGCGGGCAATGCTGTGGACAATG-3′ and 5′-CTACTAGGATCCCCGTTAACCACGTCGTAGTAGTAATTG-3′ and subcloned upstream of the citrine sequence. Rat CD45 was amplified from RBL-2H3 cDNA using the primers 5′-TAGTAGACGCGTGCCACCATGTATTTGTGGCTCAAACTTCTGGCC-3′ and 5′-CTACTAGGTACCCCTGAGCTCGGGGTTAGAGCTGGGC-3′, which was subcloned into pHR upstream of the HaloTag sequence. For the chimeric CD86CD45 and CD43CD45 forms, the transmembrane- and intracellular domain–encoding segments of rCD45 were amplified using the primers 5′-CCTCCCCCAGACCACGCACTGATTATATTCC-3′ or 5′-GAGAACTCACGAGGCGCACTGATTATATTCC-3′, respectively, to enable overlap with the extracellular domain–encoding segments of hCD86 and hCD43 amplified from cDNA prepared from RNA extracted from human THP-1 cells with the primers 5′-TAGTAGACGCGTGCCACCATGGATCCCCAGTGCACTATG-3′ and 5′-GGAATATAATCAGTGCGTGGTCTGGGGGAGG-3′ (for CD86) or 5′-TAGTAGACGCGTGCCACCATGGCCACGCTTCTCCTTCTCC-3′ and 5′-GGAATATAATCAGTGCGCCTCGTGAGTTCTC-3′ (for CD43). These fragments were used in chimeric polymerase chain reactions to generate the full-length constructs, which were then inserted into pHR upstream of HaloTag. For all fluorescent constructs, human embryonic kidney (HEK) 293 cells were cotransfected with pHR vectors and with pMD.G and p8.91 with GeneJuice (Novagen) as per the manufacturer’s instructions. Harvested lentivirus was incubated with RBL-2H3 cells in culture for 72 hours before expression of the relevant proteins was assessed by flow cytometry. A pure population of cells expressing all constructs was then derived by fluorescence-activated cell sorting.

注意:以上内容是从某篇研究文章中自动提取的,可能无法正确显示。



Q&A
请登录并在线提交您的问题
您的问题将发布在Bio-101网站上。我们会将您的问题发送给本研究方案的作者和具有相关研究经验的Bio-protocol成员。我们将通过您的Bio-protocol帐户绑定邮箱进行消息通知。