Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2002-05-08
2004-10-26
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091510, C435S320100, C435S325000, C435S252330, C435S489000
Reexamination Certificate
active
06808906
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of molecular biology and in particular to the creation and use of gene libraries containing cloned cDNAs that encode expressed genes.
BACKGROUND OF THE INVENTION
A common practice in molecular biology is to create “gene libraries,” which are collections of cloned fragments of DNA that represent genetic information in an organism, tissue or cell type. To construct a library, desired DNA fragments are prepared and inserted by molecular techniques into self-replicating units generally called cloning vectors. Each DNA fragment is therefore represented as part of an individual molecule, which can be reproduced in a single bacterial colony or bacteriophage plaque. Individual clones of interest can be identified by various screening methods, and then grown and purified in large quantities to allow study of gene organization, structure and function.
Only a small fraction of the genetic information for an organism is actually used in an individual cell or tissue at a particular time. A cDNA library is a type of gene library in which only DNA for actively expressed genes is cloned. These active genes can be selectively cloned over silent genes because the DNA for active genes is transcribed into messenger RNA (mRNA) as part of the pathway by which proteins are made. RNA molecules are polar in nature, i.e. the constituent nucleoside bases are linked via phosphodiester bonds between the 3′ ribosyl position of one nucleoside and the 5′ ribosyl position on the following nucleoside. RNA is synthesized in the 5′ to 3′ direction, and mRNAs are read by ribosomes in the same direction, such that proteins are synthesized from N-terminus to C-terminus. Over the past decade, cDNA libraries have become the standard source from which thousands of genes have been isolated for further study.
cDNA libraries may be expression libraries, whereby the cDNAs are transcribed and translated, resulting in the production of polypeptides corresponding to mRNA-encoded proteins. The activity of cDNA expression products may be assayed, and the function of corresponding mRNAs and proteins encoded thereby may be determined.
Full length cDNA, which comprises the entire open reading frame (ORF) of an mRNA, is desirable for many applications. Alternatively, partial cDNA and cDNA fragments are useful in some applications, for example, identifying functional domains within proteins. Interestingly, microdomains can exert unique biological effects compared to the parental molecules from which they are derived (Lorens et. al., Mol. Therapy, 1:438-447, 2000). The ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention.
The use of retroviruses is desirable for the stable transduction of genetic material into host cells, particularly host cells which are poorly transfectable, such as myoblasts and lymphocytes.
One object of the present invention is to provide methods and compositions for stably expressing genetic effectors, comprising random cDNAs, in host cells.
An additional object of the invention is to provide methods and compositions to screen for genetic effectors, comprising random cDNAs, that alter cell phenotype in a desirable way.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for producing directional random cDNA libraries. Directional random cDNA libraries comprising pluralities of directional random cDNA expression vectors, and methods of using these libraries, are also provided.
In one aspect of the invention, directional random cDNA expression vector libraries are provided. Each library comprises a plurality of directional random cDNA expression vectors. In a preferred embodiment, libraries comprising expression vectors with random cDNA in sense orientation are provided. In another embodiment, libraries comprising expression vectors with random cDNA in antisense orientation are provided. In another embodiment, libraries comprising a mixture of expression vectors with random cDNAs in sense orientation and antisense orientation are provided. As discussed below, the methods provided herein for making random cDNA libraries involve the directional cloning of random cDNAs into expression vectors. Accordingly, the orientation of a random cDNA in each vector is predetermined, facilitating construction of sense libraries, antisense libraries, and mixtures thereof. Such a scheme provides for the expression of antisense nucleic acid and nucleic acid corresponding in sequence to mRNA, as desired.
It will be understood that the cDNA libraries of the present invention comprise vectors, which comprise random cDNAs, which random cDNAs are directionally positioned in expression vectors in sense orientation, or antisense orientation. These libraries are sometimes referred to herein as directional random cDNA libraries. For the ease of description, the terms “directional” and “random” will often be omitted when referring herein to these libraries and methods of making the same.
In a preferred embodiment, the present invention provides cDNA expression vector libraries, each comprising a plurality of expression vectors, each vector comprising a) a first nucleic acid comprising a cDNA; b) a second nucleic acid which is a fusion partner; and c) a transcriptional regulatory sequence recognized by a host cell, wherein the first and second nucleic acids form a fusion nucleic acid which is operably linked to the transcriptional regulatory region (sometimes referred to herein as a transcriptional regulatory sequence). In some embodiments, the vectors also comprise a translational regulatory region (sometimes referred to herein as a translational regulatory sequence or start site) which forms part of the fusion nucleic acid and initiates translation of the fusion nucleic acid.
Preferred cDNAs for use in the present invention comprise sequences complementary to complete or near complete 5′ mRNA ends, including native translational start sites, which facilitate translation of cDNA encoded transcript in a host cell.
Other cDNAs may be used however, as will be appreciated by those in the art. For example, cDNAs lacking native translation start sequences, and comprising sequences complementary to 3′ mRNA ends also find use in some embodiments of the present invention.
In a preferred embodiment, the fusion partner encodes a detectable protein. In a preferred embodiment, the detectable protein is an autofluorescent protein. In a further preferred embodiment, the autofluorescent protein is a green fluorescent protein (GFP). In a further preferred embodiment, the autofluorescent protein is a GFP from Aequorea, or one of the well known variants thereof including red flourescent protein (RFP), blue fluorescent protein (BFP), and yellow fluorescent protein (YFP). In another further preferred embodiment, the autofluorescent protein is a GFP from Renilla. In another further preferred embodiment, the autofluorescent protein is a GFP from Ptilosarcus. In another preferred embodiment, the autofluorescent protein is a GFP homologue from Anthozoa species (Matz et al., Nat. Biotech., 17:969-973, 1999).
In a preferred embodiment, the first nucleic acid is fused to the 5′ end of the second nucleic acid. The expression products of such a vector include a fusion nucleic acid wherein cDNA encoded sequence is located at the 5′ end and nucleic acid sequence encoding detectable protein is located at the 3′ end. Expression products also include a fusion protein that comprises an N-terminal polypeptide encoded by cDNA and a C-terminal polypeptide which is a detectable protein moiety. In embodiments where cDNA is inserted in antisense orientation, the expression products include a fusion nucleic acid wherein antisense nucleic acid is located at the 5′ end and nucleic acid sequence encoding detectable protein is located at the 3′ end.
In a preferred embodiment, the expression vector does not co
Payan Donald
Shen Mary
Wu Xian
Yu Simon
Diehl James J.
Francis Carol L.
Keddie James S.
Ketter James
Rigel Pharmaceuticals Inc.
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