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Bi-directionally cloned random cdna expression vector libraries, compositions and methods of useRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding AssayBi-directionally cloned random cdna expression vector libraries, compositions and methods of use description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070166769, Bi-directionally cloned random cdna expression vector libraries, compositions and methods of use. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] 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 [0002] 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. [0003] 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. [0004] 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. [0005] 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 domains within proteins, and for identifying genetic effectors having desirable activity. 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. [0006] 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. [0007] One object of the present invention is to provide methods and compositions for stably expressing genetic effectors, comprising random cDNAs, in host cells. [0008] 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 [0009] The present invention provides methods and compositions for producing bi-directional random cDNA libraries. Bi-directional random cDNA libraries comprising pluralities of random cDNA expression vectors, which plurality is a mixture of vectors having cDNAs in sense and antisense orientation, are also provided. In a preferred embodiment, the random cDNA expression vectors provided herein comprise random cDNA fragments. Methods of using these libraries are also provided. [0010] In one aspect of the invention, bi-directional random cDNA expression vector libraries are provided. Each library comprises a plurality of random cDNA expression vectors. Each library further comprises three different types of random cDNA expression vectors which differ in the orientation and translational frame of the cDNA inserts therein and in the expression products they produce. In the first vector type, a random cDNA is operably linked to transcriptional and translational regulatory sequences in sense orientation and in frame. In the second vector type, a random cDNA is operably linked to transcriptional and translational regulatory sequences in sense orientation and out of frame. In the third vector type, a random cDNA is operably linked to transcriptional regulatory sequence in antisense orientation. [0011] Methods for synthesizing bi-directional random cDNA expression vector libraries comprising the three different types of vectors are also provided herein. An important, desirable feature of these methods is that separate synthesis steps are not required to produce these three different types of random cDNA expression vectors. [0012] It will be understood that the cDNA libraries of the present invention comprise vectors, which comprise random cDNAs, which random cDNAs are positioned in expression vectors in sense or antisense orientation (bi-directional). These libraries are sometimes referred to herein as bi-directional random cDNA libraries. For the ease of description, the terms "bi-directional" and "random" will often be omitted when referring herein to these libraries and methods of making the same. [0013] In a preferred embodiment, the expression vector library comprises 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). The vectors also comprise a translational regulatory region (sometimes referred to herein as a translational regulatory sequence) which forms part of the fusion nucleic acid and initiates translation of the fusion nucleic acid. [0014] In a preferred embodiment, the cDNA is a cDNA restriction fragment, preferably between about 0.2 and about 2.0 kb in size. [0015] 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). [0016] In a preferred embodiment, the first nucleic acid is fused to the 3' 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 3' end and nucleic acid sequence encoding detectable protein is located at the 5' end. Expression products also include a fusion protein that comprises a C-terminal polypeptide encoded by cDNA and an N-terminal polypeptide which is a detectable protein moiety. In a library comprising such, vectors, some cDNAs will translate in frame while others will translate out of frame, encoding what are herein referred to as "random peptides". As cDNA is also inserted in antisense orientation, the expression products include fusion nucleic acids wherein antisense nucleic acid is located at the 3' end and nucleic acid sequence encoding detectable protein is located at the 5' end. The expression products also include fusion proteins that comprise C-terminal polypeptide encoded by an antisense cDNA transcript, also referred to herein as "random peptide", and an N-terminal polypeptide which is a detectable protein moiety. [0017] The libraries provided herein comprise mixtures of vectors having cDNAs in sense or antisense orientation. cDNAs in sense orientation in the expression vectors provided herein may be translated in frame or out of frame, as discussed further below. In addition, cDNAs in antsense orientation may also be translated. Accordingly, internal "stop" codons (TAA, TGA, TAG) may be encountered, interrupting or inhibiting translation. For clarity of description, the occurrence of internal translational "stop" codons within antisense cDNAs and cDNAs translated out of frame is not treated in every embodiment discussed herein, though it is understood that such "stop" codons may occur. [0018] In another 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 libraries comprising such vectors, some cDNAs will translate in frame while others will translate out of frame as random peptides. As cDNA is also inserted in antisense orientation, the expression products include fusion nucleic acids wherein antsense nucleic acid is located at the 5' end and nucleic acid sequence encoding detectable protein is located at the 3' end. The expression products also include fusion proteins that comprise N-terminal polypeptide encoded by an antsense cDNA transcript (random peptide) and a C-terminal polypeptide which is a detectable protein moiety. [0019] In another embodiment, the first nucleic acid is positioned within the second nucleic acid (e.g., the second nucleic acid comprises the first nucleic acid). Expression products of such vectors include fusion nucleic acids wherein cDNA-encoded sequence is located within nucleic acid sequence encoding detectable protein. Expression products also include fusion proteins that comprise cDNA-encoded peptides within detectable proteins, preferably in the surface exposed loop region of a detectable protein, as described herein. In libraries comprising such vectors, some cDNAs will translate in frame while others will translate out of frame as random peptides. As cDNA is also inserted in antisense orientation, the expression products include fusion nucleic acids wherein antisense nucleic acid is located within nucleic acid sequence encoding detectable protein. The expression products also include fusion proteins that comprise antisense cDNA-encoded peptides (random peptides) within detectable proteins. [0020] In a preferred embodiment, the expression vector additionally comprises a third nucleic acid sequence, referred to herein as a linker, which is interposed between the first and second nucleic acids. In this embodiment, the linker may encode a linking peptide that joins cDNA encoded peptide to the detectable protein moiety in a fusion protein. Alternatively, the linker may be a separation sequence that provides for the expression of separate cDNA encoded peptide and detectable protein moieties. [0021] In a preferred embodiment, the linker encodes a peptide linker that joins cDNA encoded peptide to the detectable protein moiety in a fusion protein. Such a linker may be used to fuse the first nucleic acid to the 5' end or the 3' end of the second nucleic acid. Preferably, cDNA-encoded peptide is C-terminal to the detectable protein moiety in the fusion protein. Preferably, the detectable protein is GFP. Preferred linkers are rich in the amino acids glycine and serine, as described herein, and are from about 20 to about 30, more preferably about 25 to about 28 amino acids in length. Continue reading about Bi-directionally cloned random cdna expression vector libraries, compositions and methods of use... 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