US6255071B1
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Mammalian viral vectors and their uses
Assignee: Cold Spring Harbor Laboratory
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Filing: 1997-03-19
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Granted: 2001-07-03
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Expires: 2017-03-19
Abstract
Abstract The present invention relates to methods and compositions for the elucidation of mammalian gene function. Specifically, the present invention relates to methods and compositions for improved mammalian complementation screening, functional inactivation of specific essential or non-essential mammalian genes, and identification of mammalian genes which are modulated in response to specific stimuli.In particular, the compositions of the present invention include, but are not limited to, replication-deficient retroviral vectors, libraries comprising such vectors, retroviral particles produced by such vectors in conjunction with retroviral packaging cell lines, integrated provirus sequences derived from the retroviral particles of the invention and circularized provirus sequences which have been excised from the integrated provirus sequences of the invention. The compositions of the present invention further include novel retroviral packaging cell lines.
Inventors
- 👤 David H. Beach
- 👤 Gregory J. Hannon
- 👤 Douglas Conklin
- 👤 Peiqing Sun
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 17:52
CPC Classifications
C; C40; C40B; C40B40/00; C40B40/02; C07; C07K; C07K14/00; C07K14/005; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/1037; C12N15/63; C12N15/79; C12N15/85; C12N15/86; C12N2710/00; C12N2710/00011; C12N2710/20011; C12N2710/20022; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/13011; C12N2740/13041; C12N2740/13043; C12N2800/00; C12N2800/10; C12N2800/108; C12N2800/30; C12N2800/60; C12N2840/00; C12N2840/20; C12N2840/203; C12N2840/206
Claims Analysis
Total Claims: 59
Claim 1
Independent Claim
79 words
A method for expressing a heterologous nucleic acid sequence in a mammalian cell, comprising introducing, into chromosomal DNA of mammalian cells isolated in culture, a retroviral vector comprising: (i) a 3′ LTR sequence for integration of the vector into chromosomal DNA of the mammalian cell; (ii) a heterologous nucleic acid sequence to be expressed in the mammalian cell; (iii) proviral excision elements, contained within the 3′ LTR sequence, for excising a proviral form of the vector from chromosomal DNA.
Claim 2
Independent Claim
79 words
A method for expressing a heterologous nucleic acid sequence in a mammalian cell, comprising introducing, into chromosomal DNA of mammalian cells isolated in culture, a retroviral vector comprising: (i) a 3′ LTR sequence for integration of the vector into chromosomal DNA of the mammalian cell; (ii) a heterologous nucleic acid sequence to be expressed in the mammalian cell; (iii) proviral excision elements, contained within the 3′ LTR sequence, for excising a proviral form of the vector from chromosomal DNA.
Claim 3
Dependent Claim
30 words
The method of claim 1 , further comprising a step of detecting a change in phenotype of the mammalian cell as a result of expression of the heterologous nucleic acid.
Claim 4
Dependent Claim
20 words
The method of claim 2 , wherein the change in phenotype includes a change in growth of the mammalian cell.
Claim 5
Dependent Claim
20 words
The method of claim 2 , wherein the change in phenotype includes a change in differentiation of the mammalian cell.
Claim 6
Dependent Claim
25 words
The method of claim 2 , wherein the change in phenotype includes a change in expression of one or more genes of the mammalian cell.
Claim 7
Dependent Claim
24 words
The method of claim 2 , wherein the change in phenotype includes a loss-of-function of a gene or gene product of the mammalian cell.
Claim 8
Dependent Claim
27 words
The method of claim 2 , wherein the change in phenotype includes a change in the level of expression of a secreted protein or cell surface protein.
Claim 9
Dependent Claim
33 words
The method of claim 1 , wherein the mammalian cells are infected with a library of the retroviral vectors which is diverse with respect to the sequence of the heterologous nucleic acid sequence.
Claim 10
Dependent Claim
21 words
The method of claim 1 or 8 , wherein the heterologous nucleic acid sequence includes a coding sequence for a polypeptide.
Claim 11
Dependent Claim
14 words
The method of claim 9 , wherein the coding sequence encodes an intracellular polypeptide.
Claim 12
Dependent Claim
17 words
The method of claim 9 , wherein the coding sequence encodes a secreted or cell surface polypeptide.
Claim 13
Dependent Claim
25 words
The method of claim 1 or 8 , wherein the heterologous nucleic acid sequence comprises a cDNA or genomic DNA coding sequence for a polypeptide.
Claim 14
Dependent Claim
19 words
The method of claim 1 or 8 , wherein the heterologous nucleic acid sequence includes a genetic suppressor element.
Claim 15
Dependent Claim
36 words
The method of claim 13 , wherein the genetic suppressor element is selected from the group consisting of an antisense construct, a coding sequence for a dominant negative mutant or fragment of protein, and a ribozyme.
Claim 16
Dependent Claim
21 words
The method of claim 1 or 8 , wherein the heterologous nucleic acid sequence includes a coding sequence for a peptide.
Claim 17
Dependent Claim
67 words
The method of claim 8 , wherein the heterologous nucleic acid sequence includes a coding sequence for a fusion protein comprising a first polypeptide portion encoded by a cloned gene sequence and a second polypeptide portion which is detectable upon secretion of the fusion protein from the mammalian cell, the library of retroviral vectors being diverse with respect to the coding sequence for the first polypeptide portion.
Claim 18
Dependent Claim
13 words
The method of claim 1 , wherein the vector is a replication-deficient virus.
Claim 19
Dependent Claim
25 words
The method of claim 1 , wherein the vector further includes a proviral recovery element for isolating the vector from a mixture of nucleic acids.
Claim 20
Dependent Claim
15 words
The method of claim 1 , wherein the excision elements comprise enzyme-assisted site-specific integration sequences.
Claim 21
Dependent Claim
14 words
The method of claim 19 , wherein the excision elements include recombinase target sites.
Claim 22
Dependent Claim
20 words
The method of claim 20 , wherein the recombinase target sites are target sites for Cre recombinase or Flp recombinase.
Claim 23
Dependent Claim
14 words
The method of claim 1 , wherein the excision elements include restriction enzyme sites.
Claim 24
Dependent Claim
40 words
The method of claim 1 , wherein the excision elements are positioned in the vector such that, upon excision of the vector from chromosomal DNA, the excised vector can be used directly to generate virus for subsequent rounds of infection.
Claim 25
Dependent Claim
23 words
The method of claim 1 , wherein the vector further includes a packaging signal for packaging the vector in an infectious viral particle.
Claim 26
Dependent Claim
26 words
The method of claim 1 , wherein the vector further comprises a polycistronic message cassette for transcribing the heterologous nucleic acid sequence as a polycistronic message.
Claim 27
Dependent Claim
56 words
The method of claim 25 , wherein the polycistronic message cassette comprises the heterologous nucleic acid sequence, or the restriction cloning sites for cloning the heterologous nucleic acid sequence, disposed in the vector proximal to one or more marker genes such that the heterologous nucleic acid sequence and marker gene(s) are transcribed as a polycistronic message.
Claim 28
Dependent Claim
22 words
The method of claim 25 , wherein the polycistronic message includes internal ribosome entry sites (IRES) between coding sequences of the message.
Claim 29
Dependent Claim
23 words
The method of claim 18 , wherein the proviral recovery element comprises a nucleic acid sequence specifically bound by a DNA binding polypeptide.
Claim 30
Dependent Claim
35 words
The method of claim 20 , wherein the vector further comprises at least one bacterial origin of replication disposed in the vector such that, upon excision, the origin of replication is present in the provirus.
Claim 31
Dependent Claim
16 words
The method of claim 29 , wherein the bacterial origin of replication is a non-pUC ori.
Claim 32
Dependent Claim
18 words
The method of claim 29 , wherein the bacterial origin of replication is a single-stranded origin of replication.
Claim 33
Dependent Claim
24 words
The method of claim 31 , wherein the bacterial origin of replication is selected from the group consisting of RK2 OriV and f1phage Ori.
Claim 34
Dependent Claim
16 words
The method of claim 1 , wherein the vector further comprises a selectable bactcrial marker gene.
Claim 35
Dependent Claim
26 words
The method of claim 31 , wherein the selectable bacterial marker gene renders a bacterial host cell resistant to a drug or complements a cellular phenotype.
Claim 36
Dependent Claim
36 words
The method of claim 33 , wherein the selectable bacterial marker gene renders a bacterial host cell resistant to a drug selected from the group consisting of kanamycin/G418, zeocin, actinomycin, ampicillin, gentamycin, tetracycline, chloramphenicol and penicillin.
Claim 37
Dependent Claim
27 words
The method of claim 1 , wherein the vector further comprises a mammalian marker gene, the expression of which provides a detectable phenotype in a host cell.
Claim 38
Dependent Claim
26 words
The method of claim 36 , wherein expression of the mammalian marker gene renders the host cell resistant to a drug or complements a cellular phenotype.
Claim 39
Dependent Claim
23 words
The method of claim 36 , wherein the mammalian marker gene encodes a protein providing resistance to kanamycin/G418, hygromycin, mycophenolic acid or neomycin.
Claim 40
Dependent Claim
27 words
The method of claim 36 , wherein the mammalian marker gene encodes a fluorescent protein, or an enzyme which can alter the fluorescence of the host cell.
Claim 41
Dependent Claim
16 words
The method of claim 39 , wherein the mammalian marker gene encodes a green fluorescent protein.
Claim 42
Dependent Claim
50 words
The method of claim 1 , wherein the vector further comprises a lethal stutter fragment, the expression of which provides a detectable phenotype in the host cell, the expression of the lethal stuffer fragment being dependent on the presence or absence in the vector of the heterologous nucleic acid sequence.
Claim 43
Dependent Claim
28 words
The method of claim 1 , wherein the vector further comprises a constitutive transcriptional regulatory sequence for regulating transcription of the heterologous nucleic acid in the host cell.
Claim 44
Dependent Claim
28 words
The method of claim 1 , wherein the vector further comprises an inducible transcriptional regulatory sequence for regulating transcription of the heterologous nucleic acid in the host cell.
Claim 45
Dependent Claim
15 words
The method of claim 1 , wherein the vector is incorporated in an artificial chromosome.
Claim 46
Dependent Claim
29 words
The method of claim 1 , wherein the retroviral vector is derived from a replication-deficient retrovirus lacking all or a portion of the retroviral gag, pot and/or env genes.
Claim 47
Dependent Claim
14 words
The method of claim 1 , wherein the retroviral vector is derived from pBABE.
Claim 48
Dependent Claim
13 words
The method of claim 1 , wherein the vector includes a self-inactivating LTR.
Claim 49
Dependent Claim
15 words
The method of claim 1 , wherein the vector is a closed circular nucleic acid.
Claim 50
Dependent Claim
14 words
The method of claim 1 , wherein the vector is a linear nucleic acid.
Claim 51
Dependent Claim
11 words
The method of claim 1 , wherein the vector is DNA.
Claim 52
Dependent Claim
11 words
The method of claim 1 , wherein the vector is RNA.
Claim 53
Dependent Claim
15 words
The method of claim 1 , wherein the vector is packaged in a viral particle.
Claim 54
Dependent Claim
23 words
The method of claim 8 , wherein the library of retroviral vectors includes, as the heterologous nucleic acid sequences, a normalized cDNA library.
Claim 55
Dependent Claim
28 words
The method of claim 8 , wherein the library of retroviral vectors includes, as the heterologous nucleic acid sequences, a population of coding sequences for a peptide library.
Claim 56
Dependent Claim
15 words
The method of claim 54 , wherein the peptide library is a constrained peptide library.
Claim 57
Dependent Claim
31 words
The method of claim 18 , wherein the provirus recovery element is a lac operator nucleic acid sequence, a tet operator nucleic acid sequence or a lambda operator nucleic acid sequence.
Claim 58
Dependent Claim
22 words
The method of claim 28 , wherein the DNA binding polypeptide is a lac repressor, a tet repressor or a lambda repressor.
Claim 59
Dependent Claim
12 words
The method of claim 23 , wherein the excised provirus is circularized.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2004-07-02 | 2005-01-01 | 2005-07-03 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2008-07-02 | 2009-01-01 | 2009-07-03 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2012-07-02 | 2013-01-01 | 2013-07-03 | $7700.00 | Window Closed / Lapsed |