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An internal ribosome binding site can be used to select for homologous recombinants at an immunoglobulin heavy-chain locus.

作者信息

Wood C R, Morris G E, Alderman E M, Fouser L, Kaufman R J

机构信息

Genetics Institute, Cambridge, MA 02140.

出版信息

Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8006-10. doi: 10.1073/pnas.88.18.8006.

DOI:10.1073/pnas.88.18.8006
PMID:1654552
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC52434/
Abstract

The encephalomyocarditis virus (EMCV) leader sequence is responsible for efficient, cap-independent translation initiation from the viral RNA. It has been used to increase the expression of internal coding regions on polycistronic mRNA encoded by recombinant DNA constructs. We have designed a sequence-replacement-type vector for targeting to immunoglobulin heavy-chain loci in hybridoma cells. Homologous recombination of this vector introduces a human gamma 1 constant-region sequence linked to the EMCV leader and a neomycin phosphotransferase (neo) gene. The resulting cells express a bicistronic mRNA encoding at the 5' end a chimeric murine VDJH-human C gamma 1 heavy chain, followed by neo linked to the internal ribosome binding site provided by the EMCV leader. These homologous recombinants express the chimeric heavy chain at levels equivalent to the heavy chain in the parental hybridoma. This strategy of using an EMCV-neo cassette to obtain efficient selectable marker gene expression has potential application to a range of gene targeting vectors.

摘要
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c84f/52434/a07f5b809be5/pnas01068-0115-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c84f/52434/948c67c59e2b/pnas01068-0114-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c84f/52434/66aa83c023dc/pnas01068-0115-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c84f/52434/a07f5b809be5/pnas01068-0115-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c84f/52434/948c67c59e2b/pnas01068-0114-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c84f/52434/66aa83c023dc/pnas01068-0115-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c84f/52434/a07f5b809be5/pnas01068-0115-b.jpg

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2
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引用本文的文献

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2
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Mol Gen Genet. 1994 Mar;242(5):528-38. doi: 10.1007/BF00285276.
3
Unaltered immunoglobulin expression in hybridoma cells modified by targeting of the heavy chain locus with an integration vector.

本文引用的文献

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3,3',5,5' - Tetramethylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay.3,3',5,5'-四甲基联苯胺作为辣根过氧化物酶在酶免疫测定中的Ames试验阴性显色剂。
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通过用整合载体靶向重链基因座修饰的杂交瘤细胞中未改变的免疫球蛋白表达。
Immunology. 1995 Jan;84(1):159-63.
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The sequence context of the initiation codon in the encephalomyocarditis virus leader modulates efficiency of internal translation initiation.脑心肌炎病毒前导序列中起始密码子的序列上下文调节内部翻译起始的效率。
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人类免疫球蛋白Cγ1基因的核苷酸序列。
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5
Recombinant antibodies possessing novel effector functions.具有新型效应子功能的重组抗体。
Nature. 1984;312(5995):604-8. doi: 10.1038/312604a0.
6
Introduction of a mu immunoglobulin gene into the mouse germ line: specific expression in lymphoid cells and synthesis of functional antibody.将μ免疫球蛋白基因导入小鼠种系：在淋巴细胞中的特异性表达及功能性抗体的合成。
Cell. 1984 Oct;38(3):647-58. doi: 10.1016/0092-8674(84)90259-9.
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Introduction of homologous DNA sequences into mammalian cells induces mutations in the cognate gene.将同源DNA序列导入哺乳动物细胞会诱导同源基因发生突变。
Nature. 1986;324(6092):34-8. doi: 10.1038/324034a0.
8
Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies.使用一组匹配的嵌合抗体比较人免疫球蛋白的效应器功能。
J Exp Med. 1987 Nov 1;166(5):1351-61. doi: 10.1084/jem.166.5.1351.
9
Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes.小鼠胚胎衍生干细胞中原癌基因int-2的破坏：一种将突变靶向非选择基因的通用策略。
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10
Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination.通过同源重组将DNA序列插入人类染色体β-珠蛋白基因座。
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