Patent US6025192A successfully refreshed from Google Patents.
US6025192A
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Modified retroviral vectors
Assignee: Cold Spring Harbor Laboratory
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Filing: 1996-09-20
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Granted: 2000-02-15
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Expires: 2016-09-20
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 Beach
- 👤 Gregory J. Hannon
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 23:17
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; C12N2800/30; C12N2800/60; C12N2840/00; C12N2840/20; C12N2840/203; C12N2840/206
Claims Analysis
Total Claims: 61
Claim 1
Independent Claim
72 words
A retroviral vector comprising: (a) a 3' LTR sequence for integration of the vector into chromosomal DNA of a host cell; (b) a heterologous nucleic acid sequence to be transcribed in the host cell, or one or more restriction cloning sites for cloning the heterologous nucleic acid sequence into the vector; (c) proviral excision elements, contained within the 3' LTR sequence, for excising a proviral form of the vector from chromosomal DNA.
Claim 2
Dependent Claim
72 words
A retroviral vector comprising: (a) a 3' LTR sequence for integration of the vector into chromosomal DNA of a host cell; (b) a heterologous nucleic acid sequence to be transcribed in the host cell, or one or more restriction cloning sites for cloning the heterologous nucleic acid sequence into the vector; (c) proviral excision elements, contained within the 3' LTR sequence, for excising a proviral form of the vector from chromosomal DNA.
Claim 3
Dependent Claim
21 words
The vector of claim 1, wherein said heterologous nucleic acid sequence comprises a cDNA or gDNA coding sequence for a polypeptide.
Claim 4
Dependent Claim
17 words
A retroviral library comprising a multiplicity of the retroviral vectors of claim 1, having different heterologous sequences.
Claim 5
Dependent Claim
10 words
A retroviral particle including the retroviral vector of claim 1.
Claim 6
Dependent Claim
18 words
An integrated provirus derived from the retrovirus of claim 1, said provirus integrated into cells isolated in culture.
Claim 7
Dependent Claim
17 words
An excised provirus obtained by the excision of a provirus integrated by the vector of claim 1.
Claim 8
Dependent Claim
12 words
The vector of claim 1 wherein said vector is a replication-deficient virus.
Claim 9
Dependent Claim
24 words
The vector of claim 1, wherein said vector further includes a proviral recovery element for isolating the vector from a mixture of nucleic acids.
Claim 10
Dependent Claim
14 words
The vector of claim 1, wherein said excision elements comprise enzyme-assisted site-specific integration sequences.
Claim 11
Dependent Claim
13 words
The vector of claim 9, wherein said excision elements include recombinase target sites.
Claim 12
Dependent Claim
19 words
The vector of claim 10, wherein said recombinase target sites are target sites for Cre recombinase or Flp recombinase.
Claim 13
Dependent Claim
13 words
The vector of claim 1, wherein said excision elements include restriction enzyme sites.
Claim 14
Dependent Claim
39 words
The vector of claim 1, wherein said 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 15
Dependent Claim
22 words
The vector of claim 1, wherein said vector further includes a packaging signal for packaging the vector in an infectious viral particle.
Claim 16
Dependent Claim
22 words
The vector of claim 1, further comprising a polycistronic message cassette for transcribing the heterologous nucleic acid sequence as a polycistronic message.
Claim 17
Dependent Claim
55 words
The vector of claim 15, wherein said polycistronic message cassette comprises the heterologous nucleic acid sequence, or the restriction cloning sites for cloning the heterologous nucleic acid sequence, disposed in said 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 18
Dependent Claim
21 words
The vector of claim 15, wherein said polycistronic message includes internal ribosome entry sites (IRES) between coding sequences of the message.
Claim 19
Dependent Claim
22 words
The vector of claim 8, wherein said proviral recovery element comprises a nucleic acid sequence specifically bound by a DNA binding polypeptide.
Claim 20
Dependent Claim
34 words
The vector of claim 1, 25 or 28, further comprising 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 21
Dependent Claim
15 words
The vector of claim 19, wherein said bacterial origin of replication is a non-pUC ori.
Claim 22
Dependent Claim
17 words
The vector of claim 19, wherein said bacterial origin of replication is a single-stranded origin of replication.
Claim 23
Dependent Claim
24 words
The vector of claim 20, wherein said bacterial origin of replication is selected from the group consisting of RK2 OriV and f1 phage Ori.
Claim 24
Dependent Claim
12 words
The vector of claim 1, further comprising a selectable bacterial marker gene.
Claim 25
Dependent Claim
25 words
The vector of claim 23, wherein said selectable bacterial marker gene renders a bacterial host cell resistant to a drug or complements a cellular phenotype.
Claim 26
Dependent Claim
35 words
The vector of claim 23, wherein said 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 27
Dependent Claim
23 words
The vector of claim 1, further comprising a mammalian marker gene, the expression of which provides a detectable phenotype in a host cell.
Claim 28
Dependent Claim
25 words
The vector of claim 26, wherein expression of said mammalian marker gene renders the host cell resistant to a drug or complements a cellular phenotype.
Claim 29
Dependent Claim
22 words
The vector of claim 26, wherein said mammalian marker gene encodes a protein providing resistance to kanamycin/G418, hygromycin, mycophenolic acid or neomycin.
Claim 30
Dependent Claim
26 words
The vector of claim 26, wherein said mammalian marker gene encodes a fluorescent protein, or an enzyme which can alter the fluorescence of the host cell.
Claim 31
Dependent Claim
15 words
The vector of claim 29, wherein said mammalian marker gene encodes a green fluorescent protein.
Claim 32
Dependent Claim
46 words
The vector of claim 1, further comprising a lethal stuffer 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 33
Dependent Claim
21 words
The vector of claim 1, 10 or 18, wherein said heterologous nucleic acid sequence includes a coding sequence for a polypeptide.
Claim 34
Dependent Claim
13 words
The vector of claim 32, wherein said coding sequence encodes an intracellular polypeptide.
Claim 35
Dependent Claim
16 words
The vector of claim 32, wherein said coding sequence encodes a secreted or cell surface polypeptide.
Claim 36
Dependent Claim
16 words
The vector of claim 1, wherein said heterologous nucleic acid sequence includes a genetic suppressor element.
Claim 37
Dependent Claim
35 words
The vector of claim 35, wherein said 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 38
Dependent Claim
24 words
The vector of claim 1, further comprising a constitutive transcriptional regulatory sequence for regulating transcription of the heterologous nucleic acid in the host cell.
Claim 39
Dependent Claim
24 words
The vector of claim 1, further comprising an inducible transcriptional regulatory sequence for regulating transcription of the heterologous nucleic acid in the host cell.
Claim 40
Dependent Claim
14 words
The vector of claim 1, wherein said vector is incorporated in an artificial chromosome.
Claim 41
Dependent Claim
28 words
The vector of claim 1, wherein said retroviral vector is derived from a replication-deficient retrovirus lacking all or a portion of the retroviral gag, pol and/or env genes.
Claim 42
Dependent Claim
13 words
The vector of claim 1, wherein said retroviral vector is derived from pBABE.
Claim 43
Dependent Claim
26 words
The vector of claim 1, wherein said LTR sequences include said excision elements, and said excised vector can be used directly to generate packaged retroviral vectors.
Claim 44
Dependent Claim
12 words
The vector of claim 42, wherein said vector includes a self-inactivating LTR.
Claim 45
Dependent Claim
14 words
The vector of claim 1, wherein said vector is a closed circular nucleic acid.
Claim 46
Dependent Claim
13 words
The vector of claim 1, wherein said vector is a linear nucleic acid.
Claim 47
Dependent Claim
10 words
The vector of claim 1, wherein said vector is DNA.
Claim 48
Dependent Claim
10 words
The vector of claim 1, wherein said vector is DNA.
Claim 49
Dependent Claim
14 words
The vector of claim 1, wherein said vector is packaged in a viral particle.
Claim 50
Dependent Claim
26 words
A library of vectors comprising a population of vectors according to claim 1, 11 or 13, the library including vectors having different heterologous nucleic acid sequences.
Claim 51
Dependent Claim
21 words
The vector library of claim 49, wherein said library comprises, as the heterologous nucleic acid sequences, a population of cDNA sequences.
Claim 52
Dependent Claim
15 words
The vector library of claim 50, wherein said cDNA library is a normalized cDNA library.
Claim 53
Dependent Claim
25 words
The vector library of claim 49, wherein said library comprises, as the heterologous nucleic acid sequences, a population of coding sequences for a peptide library.
Claim 54
Dependent Claim
23 words
The vector library of claim 52, wherein said peptide library is a constrained peptide library, as for example, part of a fusion protein.
Claim 55
Dependent Claim
22 words
The vector library of claim 49, wherein said library comprises, as the heterologous nucleic acid sequences, a population of genetic suppressor elements.
Claim 56
Dependent Claim
29 words
The vector library of claim 54, wherein said genetic suppressor elements are selected from the group consisting of an antisense constructs, coding sequences for fragments of proteins and ribozymes.
Claim 57
Dependent Claim
30 words
The vector of claim 8, wherein said 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
21 words
The vector of claim 18, wherein said DNA binding polypeptide is a lac repressor, a tet repressor or a lambda repressor.
Claim 59
Dependent Claim
16 words
The retroviral vector library of claim 3, wherein said cDNA library is a normalized cDNA library.
Claim 60
Dependent Claim
13 words
The retroviral library of claim 3, wherein said library is a single-stranded library.
Claim 61
Dependent Claim
12 words
The excised provirus of claim 6, wherein said excised provirus is circularized.
References Cited (Backward Citations)
US4959317A
Cited
US5378618A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2003-02-14 | 2003-08-16 | 2004-02-15 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2007-02-14 | 2007-08-16 | 2008-02-15 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2011-02-14 | 2011-08-16 | 2012-02-15 | $7700.00 | Window Closed / Lapsed |