U.S. patent application number 10/746207 was filed with the patent office on 2005-07-07 for cells and non-human organisms containing predetermined genomic modifications and positive-negative selection methods and vectors for making same.
Invention is credited to Capecchi, Mario R., Thomas, Kirk R..
Application Number | 20050149998 10/746207 |
Document ID | / |
Family ID | 30773727 |
Filed Date | 2005-07-07 |
United States Patent
Application |
20050149998 |
Kind Code |
A1 |
Capecchi, Mario R. ; et
al. |
July 7, 2005 |
Cells and non-human organisms containing predetermined genomic
modifications and positive-negative selection methods and vectors
for making same
Abstract
Positive-negative selector (PNS) vectors are provided for
modifying a target DNA sequence contained in the genome of a target
cell capable of homologous recombination. The vector comprises a
first DNA sequence which contains at least one sequence portion
which is substantially homologous to a portion of a first region of
a target DNA sequence. The vector also includes a second DNA
sequence containing at least one sequence portion which is
substantially homologous to another portion of a second region of a
target DNA sequence. A third DNA sequence is positioned between the
first and second DNA sequences and encodes a positive selection
marker which when expressed is functional in the target cell in
which the vector is used. A fourth DNA sequence encoding a negative
selection marker, also functional in the target cell, is positioned
5' to the first or 3' to the second DNA sequence and is
substantially incapable of homologous recombination with the target
DNA sequence. The invention also includes transformed cells
containing at least one predetermined modification of a target DNA
sequence contained in the genome of the cell. In addition, the
invention includes organisms such as non-human transgenic animals
and plants which contain cells having predetermined modifications
of a target DNA sequence in the genome of the organism.
Inventors: |
Capecchi, Mario R.; (Salt
Lake City, UT) ; Thomas, Kirk R.; (Salt Lake City,
UT) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
30773727 |
Appl. No.: |
10/746207 |
Filed: |
December 23, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10746207 |
Dec 23, 2003 |
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09724962 |
Nov 28, 2000 |
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6689610 |
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09724962 |
Nov 28, 2000 |
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08781559 |
Jan 9, 1997 |
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6204061 |
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08781559 |
Jan 9, 1997 |
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08461827 |
Jun 5, 1995 |
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5627059 |
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08461827 |
Jun 5, 1995 |
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08084741 |
Jun 28, 1993 |
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5487992 |
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08084741 |
Jun 28, 1993 |
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08014083 |
Feb 4, 1993 |
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5464764 |
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08014083 |
Feb 4, 1993 |
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07397707 |
Aug 22, 1989 |
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Current U.S.
Class: |
800/14 ;
435/320.1; 435/325; 435/455 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 9/1077 20130101; A01K 2217/05 20130101; C12N 15/8209 20130101;
C12N 15/8213 20130101; C07K 14/755 20130101; C12N 15/907 20130101;
C07K 14/50 20130101; C07K 14/47 20130101 |
Class at
Publication: |
800/014 ;
435/325; 435/455; 435/320.1 |
International
Class: |
A01K 067/027; C12N
015/85 |
Claims
1-90. (canceled)
91. An isolated mouse embryonic stem cell having a genome
comprising a positive selection marker DNA sequence inserted into a
non-selectable gene by a homologous recombination event between
said genome and a targeting vector, thereby inactivating said gene,
wherein said gene is inactivated without a duplication of genomic
DNA sequence by said event and wherein said genome is free of an
additional copy of the positive selection marker DNA sequence
integrated by a random recombination event.
92. The isolated embryonic stem cell of claim 91 wherein said
non-selectable gene is a gene of the immune system.
93. The isolated embryonic stem cell of claim 92, wherein said
genome further comprises a heterologous protein coding sequence
other than a positive selection marker DNA sequence.
94. The isolated embryonic stem cell of claim 93, wherein said
heterologous protein coding sequence is a human gene.
95. The isolated embryonic stem cell of claim 94, wherein said
human gene is a gene of the human immune system.
96. The isolated embryonic stem cell of claim 95, wherein said
genome is without a positive screening marker DNA sequence.
97. The isolated embryonic stem cell of claim 96, wherein said
genome is without a bacterial tRNA suppressor gene.
98. An isolated mouse embryonic stem cell having a genome
comprising a human gene in place of a corresponding mouse gene,
wherein said human gene is inserted into said genome by a
homologous recombination event between said genome and a targeting
vector, without a duplication of genomic DNA sequence by said event
and wherein said genome is free of an additional copy of the
positive selection marker DNA sequence integrated by a random
recombination event.
99. The isolated mouse embryonic stem cell of claim 98, wherein
said human gene is a gene of the immune system.
100. The isolated embryonic stem cell of claim 91 produced by the
steps of: (a) transforming a population of cells with a
positive-negative selection (PNS) vector; and (b) identifying said
cell by selecting for cells containing said positive selection
marker and against cells containing said negative selection marker;
wherein said PNS vector comprises: (1) a first homologous vector
DNA sequence capable of homologous recombination with a first
region of said target DNA sequence; (2) a positive selection marker
DNA sequence capable of conferring a positive selection
characteristic in said cells; (3) a second homologous vector DNA
sequence capable of homologous recombination with a second region
of said target DNA sequence; and (4) a negative selection marker
DNA sequence, capable of conferring a negative selection
characteristic in said cells, but substantially incapable of
homologous recombination with said target DNA sequence; wherein the
spatial order of said sequences in said PNS vector is: said first
homologous vector DNA sequence, said positive selection marker DNA
sequence, said second homologous vector DNA sequence and said
negative selection marker DNA sequence as shown in FIG. 1; wherein
the 5'-3' orientation of said first homologous vector sequence
relative to said second homologous vector sequence is the same as
the 5'-3' orientation of said first region relative to said second
region of said target sequence; wherein the vector is capable of
modifying said target DNA sequence by homologous recombination of
said first homologous vector DNA sequence with said first region of
said target sequence and of said second homologous vector DNA
sequence with said second region of said target sequence.
101. A transgenic mouse having a genome comprising a positive
selection marker DNA sequence inserted into a non-selectable gene
by a homologous recombination event between said genome and a
targeting vector, thereby inactivating said gene, wherein said gene
is inactivated without a duplication of genomic DNA sequence by
said event and wherein said genomic DNA is free of an additional
copy of the positive selection marker DNA sequence integrated by a
random recombination event.
102. The transgenic mouse of claim 101 wherein said non-selectable
gene is a gene of the immune system.
103. The transgenic mouse of claim 102, wherein said genome further
comprises a heterologous protein coding sequence other than a
positive selection marker DNA sequence.
104. The transgenic mouse of claim 103, wherein said heterologous
protein coding sequence is a human gene.
105. The transgenic mouse of claim 104, wherein said human gene is
a gene of the human immune system.
106. The transgenic mouse of claim 105, wherein said genome is free
of a positive screening marker DNA sequence.
107. The transgenic mouse of claim 106, wherein said genome is free
of a bacterial tRNA suppressor gene.
108. A transgenic mouse having a genome comprising a human gene
replacing a corresponding mouse gene, wherein said human gene is
inserted into said genome by a homologous recombination event
between said genome and a targeting vector, without a duplication
of genomic DNA sequence by said even and wherein said genome is
free of an additional copy of the positive selection marker DNA
sequence integrated by a random recombination event
109. The transgenic mouse of claim 108, wherein said human gene is
a gene of the immune system.
110. An isolated cell having a genome comprising a positive
selection marker DNA sequence inserted into a non-selectable gene
by a homologous recombination event between said genome and a
targeting vector, thereby inactivating said gene, wherein said gene
is inactivated without a duplication of genomic DNA sequence by
said event and wherein said genome is free of an additional copy of
the positive selection marker DNA sequence integrated by a random
recombination event.
111. The cell of claim 110, selected from the group consisting of
hematopoietic, epithelial, liver, lung, bone marrow, endothelial,
mesenchymal, neural and muscle stem cells.
112. The cell of claim 111, which is a cell of a vertebrate
animal.
113. The cell of claim 112, which is a human cell.
114. The cell of claim 111, which is a bone marrow stem cell.
115. The cell of claim 112, which is a simian cell.
116. An isolated mouse embryonic stem cell having a genome
comprising a positive selection marker DNA sequence inserted into a
gene other than hprt by a homologous recombination event between
said genome and a targeting vector, thereby inactivating said gene,
wherein said gene is inactivated without a duplication of genomic
DNA sequence by said event and wherein said genome is free of an
additional copy of the positive selection marker DNA sequence
integrated by a random recombination event.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The invention relates to cells and non-human organisms
containing predetermined genomic modifications of the genetic
material contained in such cells and organisms. The invention also
relates to methods and vectors for making such modifications.
BACKGROUND OF THE INVENTION
[0002] Many unicellular and multicellular organisms have been made
containing genetic material which is not otherwise normally found
in the cell or organism. For example, bacteria, such as E. coli,
have been transformed with plasmids which encode heterologous
polypeptides, i.e., polypeptides not normally associated with that
bacterium. Such transformed cells are routinely used to express the
heterologous gene to obtain the heterologous polypeptide. Yeasts,
filamentous fungi and animal cells have also been transformed with
genes encoding heterologous polypeptides. In the case of bacteria,
heterologous genes are readily maintained by way of an extra
chromosomal element such as a plasmid. More complex cells and
organisms such as filamentous fungi, yeast and mammalian cells
typically maintain the heterologous DNA by way of integration of
the foreign DNA into the genome of the cell or organism. In the
case of mammalian cells and most multicellular organisms such
integration is most frequently random within the genome.
[0003] Transgenic animals containing heterologous genes have also
been made. For example, U.S. Pat. No. 4,736,866 discloses
transgenic non-human mammals containing activated oncogenes. Other
reports for producing transgenic animals include PTC Publication
No. WO82/04443 (rabbit .beta.-globin gene DNA fragment injected
into the pronucleus of a mouse zygote); EPO Publication No. 0 264
166 (Hepatitis B surface antigen and tPA genes under control of the
whey acid protein promotor for mammary tissue specific expression);
EPO Publication No. 0 247 494 (transgenic mice containing
heterologous genes encoding various forms of insulin); PTC
Publication No. WO88/00239 (tissue specific expression of a
transgene encoding factor IX under control of a whey protein
promotor); PTC Publication No. WO88/01648 (transgenic mammal having
mammary secretory cells incorporating a recombinant expression
system comprising a mammary lactogen-inducible regulatory region
and a structural region encoding a heterologous protein); and EPO
Publication No. 0 279 582 (tissue specific expression of
chloramphenicol acetyltrans-ferase under control of rat
.beta.-casein promotor in transgenic mice). The methods and DNA
constructs ("transgenes") used in making these transgenic animals
also result in the random integration of all or part of the
transgene into the genome of the organism. Typically, such
integration occurs in an early embryonic stage of development which
results in a mosaic transgenic animal. Subsequent generations can
be obtained, however, wherein the randomly inserted transgene is
contained in all of the somatic cells of the transgenic
animals.
[0004] Transgenic plants have also been produced. For example, U.S.
Pat. No. 4,801,540 to Hiatt, et al., discloses the transformation
of plant cells with a plant expression vector containing tomato
polygalacturonase (PG) oriented in the opposite orientation for
expression. The anti-sense RNA expressed from this gene is capable
of hybridizing with endogenous PG mRNA to suppress translation.
This inhibits production of PG and as a consequence the hydrolysis
of pectin by PG in the tomato.
[0005] While the integration of heterologous DNA into cells and
organisms is potentially useful to produce transformed cells and
organisms which are capable of expressing desired genes and/or
polypeptides, many problems are associated with such systems. A
major problem resides in the random pattern of integration of the
heterologous gene into the genome of cells derived from
multicellular organisms such as mammalian cells. This often results
in a wide variation in the level of expression of such heterologous
genes among different transformed cells. Further, random
integration of heterologous DNA into the genome may disrupt
endogenous genes which are necessary for the maturation,
differentiation and/or viability of the cells or organism. In the
case of transgenic animals, gross abnormalities are often caused by
random integration of the transgene and gross rearrangements of the
transgene and/or endogenous DNA often occur at the insertion site.
For example, a common problem associated with transgenes designed
for tissue-specific expression involves the "leakage" of expression
of the transgenes. Thus, transgenes designed for the expression and
secretion of a heterologous polypeptide in mammary secretory cells
may also be expressed in brain tissue thereby producing adverse
effects in the transgenic animal. While the reasons for transgene
"leakage" and gross rearrangements of heterologous and endogenous
DNA are not known with certainty, random integration is a potential
cause of expression leakage.
[0006] One approach to overcome problems associated with random
integration involves the use gene of targeting. This method
involves the selection for homologous recombination events between
DNA sequences residing in the genome of a cell or organism and
newly introduced DNA sequences. This provides means for
systematically altering the genome of the cell or organism.
[0007] For example, Hinnen, J. B., et al. (1978) Proc. Natl. Acad.
Sci. U.S.A., 75, 1929-1933 report homologous recombination between
a leu2.sup.+ plasmid and a leu2.sup.- gene in the yeast genome.
Successful homologous transformants were positively selected by
growth on media deficient in leucine.
[0008] For mammalian systems, several laboratories have reported
the insertion of exogenous DNA sequences into specific sites within
the mammalian genome by way of homologous recombination. For
example, Smithies, O., et al. (1985) Nature, 317, 230-234 report
the insertion of a linearized plasmid into the genome of cultured
mammalian cells near the .beta.-globin gene by homologous
recombination. The modified locus so obtained contained inserted
vector sequences containing a neomycin resistance gene and a Sup F
gene encoding an amber suppressor t-RNA positioned between the
.delta. and .beta.-globin structural genes. The homologous
insertion of this vector also resulted in the duplication of some
of the DNA sequence between the .delta. and .beta.-globin genes and
part of the .beta.-globin gene itself. Successful transformants
were selected using a neomycin related antibiotic. Since most
transformation events randomly inserted this plasmid, insertion of
this plasmid by homologous recombination did not confer a
selectable, cellular phenotype for homologous recombination
mediated transformation. A laborious screening test for identifying
predicted targeting events using plasmid rescue of the supF marker
in a phage library prepared from pools of transfected colonies was
used. Sib selection utilizing this assay identified the transformed
cells in which homologous recombination had occurred.
[0009] A significant problem encountered in detecting and isolating
cells, such as mammalian and plant cells, wherein homologous
recombination events have occurred lies in the greater propensity
for such cells to mediate non-homologous recombination. See Roth,
D. B., et al. (1985) Proc. Natl. Acad. Sci. U.S.A., 82 3355-3359;
Roth, D. B., et al. (1985), Mol. Cell. Biol., 5 2599-2607; and
Paszkowski, J., et al. (1988), EMBO J., 7, 4021-4026. In order to
identify homologous recombination events among the vast pool of
random insertions generated by non-homologous recombination, early
gene targeting experiments in mammalian cells were designed using
cell lines carrying a mutated form of either a neomycin resistance
(neo.sup.r) gene or a herpes simplex virus thymidine kinase
(HSV-tk) gene, integrated randomly into the host genome. Such
exogenous defective genes were then specifically repaired by
homologous recombination with newly introduced exogenous DNA
carrying the same gene bearing a differ nt mutation.
[0010] Productive gene targeting vents were identified by selection
for cells with the wild type phenotype either by resistance to the
drug G418 (neo.sup.r) or ability to grow in HAT medium (tk.sup.+).
See, e.g., Folger, K. R., et al. (1984), Cold Spring Harbor Symp.
Quant. Biol., 49, 123-138; Lin, F. L. et al. (1984), Cold Spring
Harbor Symp. Quant. Biol., 49, 139-149; Smithies, O., et al.
(1984), Cold Spring Harbor Symp. Quant. Biol., 49, 161-170; Smith,
A. J. H., et al. (1984), Cold Spring Harbor Symp. Quant. Biol., 49,
171-181; Thomas K. R., et al. (1986), Cell, 41, 419-428; Thomas, K.
R., et al. (1986), Nature, 324, 34-38; Doetschman, T., et al.
(1987), Nature, 330, 576-578; and Song, Kuy-Young, et al. (1987),
Proc. Natl. Acad. Sci. U.S.A., 84, 6820-6824. A similar approach
has been used in plant cells where partially deleted neomycin
resistance genes reportedly were randomly inserted into the genome
of tobacco plants. Transformation with vectors containing the
deleted sequences conferred resistance to neomycin in those plant
cells wherein homologous recombination occurred. Paszkowski, J., et
al. (1988), EMBO J., 7, 4021-4026.
[0011] A specific requirement and significant limitation to this
approach is the necessity that the targeted gene confer a positive
selection characteristic in those cells wherein homologous
recombination has occurred. In each of the above cases, a defective
exogenous positive selection marker was inserted into the genome.
Such a requirement severely limits the utility of such systems to
the detection of homologous recombination events involving inserted
selectable genes.
[0012] In a related approach, Thomas, K. R., et al. (1987), Cell,
51, 503-512, report the disruption of a selectable endogenous mouse
gene by homologous recombination. In this approach, a vector was
constructed containing a neomycin resistance gene inserted into
sequences encoding an exon of the mouse hypoxanthine phosphoribosyl
transferase (Hprt) gene. This endogenous gene was selected for two
reasons. First, the Hprt gene lies on the X-chromosome. Since
embryonic stem cells (ES cells) derived from male embryos are
hemizygous for Hprt, only a single copy of the Hprt gene need be
inactivated by homologous recombination to produce a selectable
phenotype. Second, selection procedures are available for isolating
Hprt.sup.- mutants. Cells wherein homologous recombination events
occurred could thereafter be positively selected by detecting cells
resistant to neomycin (neo.sup.R) and 6-thioguanine
(Hprt.sup.-).
[0013] A major limitation in the above methods has been the
requirement that the target ence in the genome, either endogenous
or exogenous, confer a selection characteristic to the cells in
which homologous recombination has occurred (i.e. neo.sup.R,
tk.sup.+ or Hprt.sup.-). Further, for those gene sequences which
confer a selectable phenotype upon homologous recombination (e.g.
the Hprt gene), the formation of such a selectable phenotype
requires the disruption of the endogenous gene.
[0014] The foregoing approaches to gene targeting are clearly not
applicable to many emerging technologies. See, e.g. Friedman, T.
(1989), Science, 244, 1275-1281 (human gene therapy); Gasser, C.
S., et al., Id., 1293-1299 (genetic engineering of plants); Pursel,
I. G., et al., Id. 1281-1288 (genetic engineering of livestock);
and Timberlake, W. E., et al., Id. et al., 13-13, 1312 (genetic
engineering of filamentous fungi). Such techniques are generally
not useful to isolate transformants wherein non-selectable
endogenous genes are disrupted or modified by homologous
recombination. The above methods are also of little or no use for
gene therapy because of the difficulty in selecting cells wherein
the genetic defect has been corrected by way of homologous
recombination.
[0015] Recently, several laboratories have reported the expression
of an expression-defective exogenous selection marker after
homologous integration into the genome of mammalian cells. Sedivy,
J. M., et al. (1989), Proc. Nat. Acad. Sci. U.S.A., 86, 227-231,
report targeted disruption of the hemizygous polyomavirus middle-T
antigen with a neomycin resistance gene lacking an initiation
codon. Successful transformants were selected for resistance to
G418. Jasin, N., et al. (1988), Genes and Development, 2, 1353-1363
report integration of an expression-defective gpt gene lacking the
enhancer in its SV40 early promotor into the SV40 early region of a
gene already integrated into the mammalian genome. Upon homologous
recombination, the defective gpt gene acts as a selectable
marker.
[0016] Assays for detecting homologous recombination have also
recently been reported by several laboratories.
[0017] Kim, H. S., et al. (1988), Nucl. Acid. S. Res., 16,
8887-8903, report the use of the polymerase chain reaction (PCR) to
identify the disruption of the mouse hprt gene. A similar strategy
has been used by others to identify the disruption of the Hox 1.1
gene in mouse ES cells (Zimmmer, A. P., et al. (1989), Nature, 338,
150-153) and the disruption of the En-2 gen by homologous r
combination in embryonic stem cells. (Joynar, A. L., t al. (1989),
Nature, 338, 153-156).
[0018] It is an object herein to provide methods whereby any
predetermined region of the genome of a cell or organism may be
modified and wherein such modified cells can be selected and
enriched.
[0019] It is a further object of the invention to provide novel
vectors used in practicing the above methods of the invention.
[0020] Still further, an object of the invention is to provide
transformed cells which have been modified by the methods and
vectors of the invention to contain desired mutations in specific
regions of the genome of the cell.
[0021] Further, it is an object herein to provide non-human
transgenic organisms, which contain cells having predetermined
genomic modifications.
[0022] The references discussed above are provided solely for their
disclosure prior to the filing date of the present application.
Nothing herein is to be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention.
SUMMARY OF THE INVENTION
[0023] In accordance with the above objects, positive-negative
selector (PNS) vectors are provided for modifying a target DNA
sequence contained in the genome of a target cell capable of
homologous recombination. The vector comprises a first DNA sequence
which contains at least one sequence portion which is substantially
homologous to a portion of a first region of a target DNA sequence.
The vector also includes a second DNA sequence containing at least
one sequence portion which is substantially homologous to another
portion of a second region of a target DNA sequence. A third DNA
sequence is positioned between the first and second DNA sequences
and encodes a positive selection marker which when expressed is
functional in the target cell in which the vector is used. A fourth
DNA sequence encoding a negative selection marker, also functional
in the target cell, is positioned 5' to the first or 3' to the
second DNA sequence and is substantially incapable of homologous
recombination with the target DNA sequence.
[0024] The above PNS vector containing two homologous portions and
a positive and a negative selection marker can be used in the
methods of the invention to modify target DNA sequences. In this
method, cells are first transfected with the above vector. During
this transformation, the PNS vector is most frequently randomly
integrated into the genome of the cell. In this case, substantially
all of the PNS vector containing the first, second, third and
fourth DNA sequences is inserted into the genome. However, some of
the PNS vector is integrated into the genome via homologous
recombination. When homologous recombination occurs between the
homologous portions of the first and second DNA sequences of the
PNS vector and the corresponding homologous portions of the
endogenous target DNA of the cell, the fourth DNA sequence
containing the negative selection marker is not incorporated into
the genome. This is because the negative selection marker lies
outside of the regions of homology in the endogenous target DNA
sequence. As a consequence, at least two cell populations are
formed. That cell population wherein random integration of the
vector has occurred can be selected against by way of the negative
selection marker contained in the fourth DNA sequence. This is
because random events occur by integration at the ends of linear
DNA. The other cell population wherein gene targeting has occurred
by homologous recombination are positively selected by way of the
positive selection marker contained in the third DNA sequence of
the vector. This cell population does not contain the negative
selection marker and thus survives the negative selection. The net
effect of this positive-negative selection method is to
substantially enrich for transformed cells containing a modified
target DNA sequence.
[0025] If in the above PNS vector, the third DNA sequence
containing the positive selection marker is positioned between
first and second DNA sequences corresponding to DNA sequences
encoding a portion of a polypeptide (e.g. within the exon of a
eucaryotic organism) or within a regulatory region necessary for
gene expression, homologous recombination allows for the selection
of cells wherein the gene containing such target DNA sequences is
modified such that it is non functional.
[0026] If, however, the positive selection marker contained in the
third DNA sequence of the PNS vector is positioned within an
untranslated region of the genome, e.g. within an intron in a
eucaryotic gene, modifications of the surrounding target sequence
(e.g. exons and/or regulatory regions) by way of substitution,
insertion and/or deletion of one or more nucleotides may be made
without eliminating the functional character of the target
gene.
[0027] The invention also includes transformed cells containing at
least one predetermined modification of a target DNA sequence
contained in the genome of the cell.
[0028] In addition, the invention includes organisms such as
non-human transgenic animals and plants which contain cells having
predetermined modifications of a target DNA sequence in the genome
of the organism.
[0029] Various other aspects of the invention will be apparent from
the following detailed description, appended drawings and
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 depicts the positive-negative selection (PNS) vector
of the invention and a target DNA sequence.
[0031] FIGS. 2a and 2b depict the results of gene targeting
(homologous recombination) and random integration of a PNS vector
into a genome.
[0032] FIG. 3 depicts a PNS vector containing a positive selection
marker within a sequence corresponding, in part, to an intron of a
target DNA sequence.
[0033] FIG. 4 is a graphic representation of the absolute frequency
of homologous recombination versus the amount of 100% sequence
homology in the first and second DNA sequences of the PNS vectors
of the invention.
[0034] FIGS. 5a, 5b, 5c and 5d depict the construction of a PNS
vector used to disrupt the INT-2 gene.
[0035] FIG. 6 depicts the construction of a PNS vector used to
disrupt the HOX1.4 gene.
[0036] FIGS. 7A, 7B and 7C depict the construction of a PNS vector
used to transform endothelial cells to express factor VIII.
[0037] FIG. 8 depicts a PNS vector to correct a defect in the
purine nucleoside phosphorylase gene.
[0038] FIG. 9 depicts a vector for promoterless PNS.
[0039] FIG. 10 depicts the construction of a PNS vector to target
an inducible promoter into the int-2 locus.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The positive-negative selection ("PNS") methods and vectors
of the invention are used to modify target DNA sequences in the
genome of cells capable of homologous recombination.
[0041] A schematic diagram of a PNS vector of the invention is
shown in FIG. 1. As can be seen, the PNS vector comprises four DNA
sequences. The first and second DNA sequences each contain portions
which are substantially homologous to corresponding homologous
portions in first and second regions of the targeted DNA.
Substantial homology is necessary between these portions in the PNS
vector and the target DNA to insure targeting of the PNS vector to
the appropriate region of the genome.
[0042] As used herein, a "target DNA sequence" is a predetermined
region within the genome of a cell which is targeted for
modification by the PNS vectors of the invention. Target DNA
sequences include structural genes (i.e., DNA sequences encoding
polypeptides including in the case of eucaryots, introns and
exons), regulatory sequences such as enhancers sequences, promoters
and the like and other regions within the genome of int rest. A
target DNA sequence may also be a sequence which, when targeted by
a vector has no effect on the function of the host genome.
Generally, the target DNA contains at least first and second
regions. See FIG. 1. Each region contains a homologous sequence
portion which is used to design the PNS vector of the invention. In
some instances, the target DNA sequence also includes a third and
in some cases a third and fourth region. The third and fourth
regions are substantially contiguous with the homologous portions
of the first and second region. The homologous portions of the
target DNA are homologous to sequence portions contained in the PNS
vector. The third and in some cases third and fourth regions define
genomic DNA sequences within the target DNA sequence which are not
substantially homologous to the fourth and in some cases fourth and
fifth DNA sequences of the PNS vector.
[0043] Also included in the PNS vector are third and fourth DNA
sequences which encode respectively "positive" and "negative"
selection markers. Examples of preferred positive and negative
selection markers are listed in Table I. The third DNA sequence
encoding the positive selection marker is positioned between the
first and second DNA sequences while the fourth DNA sequence
encoding the negative selection marker is positioned either 3' to
the second DNA sequences shown in FIG. 1, or 5' to the first DNA
sequence (not shown in FIG. 1). The positive and negative selection
markers are chosen such that they are functional in the cells
containing the target DNA.
[0044] Positive and/or negative selection markers are "functional"
in transformed cells if the phenotype expressed by the DNA
sequences encoding such selection markers is capable of conferring
either a positive or negative selection characteristic for the cell
expressing that DNA sequence. Thus, "positive selection" comprises
contacting cells transfected with a PNS vector with an appropriate
agent which kills or otherwise selects against cells not containing
an integrated positive selection marker. "Negative selection" on
the other hand comprises contacting cells transfected with the PNS
vector with an appropriate agent which kills or otherwise selects
against cells containing the negative selection marker. Appropriate
agents for use with specific positive and negative selection
markers and appropriate concentrations are listed in Table I. Other
positive selection markers include DNA sequences encoding membrane
bound polypeptides. Such polypeptides are well known to those
skilled in the art and contain a secretory sequence, an
extracellular domain, a transmembrane domain and an intracellular
domain. When expressed as a positive selection marker, such
polypeptides associate with the target cell membrane. Fluorescently
labelled antibodies specific for the extracellular domain may then
be used in a fluoresence activated cell sorter (FACS) to select for
cells expressing the membrane bound polypeptide. FACS selection may
occur before or after negative selection.
1TABLE I Selectable Markers for Use in PNS-Vectors Preferred
Concentration of Selective selective Gene Type Agents Agent
Organism Neo + G418 50-1000 .mu.g/ml Eukaryotes Neo + Kanamycin
5-500 .mu.g/ml Plants Hyg + Hygromycin 10-1000 .mu.g/ml Eukaryotes
hisD + Histidinol 5-500 .mu.g/ml Animals Gpt + Xanthine, 50-500
.mu.g/ml Animals Ble + Bleomycin 1-100 .mu.g/ml Plants Hprt +
Hypoxanthine 0.01-10 mM All HSV-tk - Acyclovir 1-100 .mu.M Animals
Gancyclovir 0.05-200 .mu.M Animals FIAU 0.02-100 .mu.M Animals Hprt
- 6-thioguanine 0.1-100 .mu.g/ml All Gpt - 6-thioxanthine 0.1-100
.mu.g/ml Animals Diphtheria - None None Animals toxin Ricin toxin -
None None Animals cytosine - 5-fluoro- 10-500 .mu.g/ml All
deaminase cytosine
[0045] The expression of the negative selection marker in the
fourth DNA sequence is generally under control of appropriate
regulatory sequences which render its expression in the target cell
independent of the expression of other sequences in the PNS vector
or the target DNA. The positive selection marker in the third DNA,
however, may be constructed so that it is independently expressed
(eg. when contained in an intron of the target DNA) or constructed
so that homologous recombination will place it under control of
regulatory sequences in the target DNA sequence. The strategy and
details of the expression of the positive selection marker will be
discussed in more detail hereinafter.
[0046] The positioning of the negative selection marker as being
either "5'" or "3'" is to be understood as relating to the
positioning of the negative selection marker relative to the 5' or
3' end of one of the strands of the double-stranded PNS vector.
This should be apparent from FIG. 1. The positioning of the various
DNA sequences within the PNS vector, however, does not require that
each of the four DNA sequences be transcriptionally and
translationally aligned on a single strand of the PNS vector. Thus,
for example, the first and second DNA sequences may have a 5' to 3'
orientation consistent with the 5' to 3' orientation of regions 1
and 2 in the target DNA sequence. When so aligned, the PNS vector
is a "replacement PNS vector" upon homologous recombination the
replacement PNS vector replaces the genomic DNA sequence between
the homologous portions of the target DNA with the DNA sequences
between the homologous portion of the first and second DNA
sequences of the PNS vector. Sequence replacement vectors are
preferred in practicing the invention. Alternatively, the
homologous portions of the first and second DNA sequence in the PNS
vector may be inverted relative to each other such that the
homologous portion of DNA sequence 1 corresponds 5' to 3' with the
homologous portion of region 1 of the target DNA sequence whereas
the homologous portion of DNA sequence 2 in the PNS vector has an
orientation which is 3' to 5' for the homologous portion of the
second region of the second region of the target DNA sequence. This
inverted orientation provides for and "insertion PNS vector". When
an insertion PNS vector is homologously inserted into the target
DNA sequence, the entire PNS vector is inserted into the target DNA
sequence without replacing the homologous portions in the target
DNA. The modified target DNA so obtained necessarily contains the
duplication of at least those homologous portions of the target DNA
which are contained in the PNS vector. Sequence replacement vectors
and sequence insertion vectors utilizing a positive selection
marker only are described by Thomas et al. (1987), Cell, 51,
503-512.
[0047] Similarly, the third and fourth DNA sequences may be
transcriptionally inverted relative to each other and to the
transcriptional orientation of the target DNA sequence. This is
only the case, however, when expression of the positive and/or
negative selection marker in the third and/or fourth DNA sequence
respectively is independently controlled by appropriate regulatory
sequences. When, for example a promoterless positive selection
marker is used as a third DNA sequence such that its expression is
to be placed under control of an endogenous regulatory region, such
a vector requires that the positive selection marker be positioned
so that it is in proper alignment (5' to 3' and proper reading
frame) with the transcriptional orientation and sequence of the
endogenous regulatory region.
[0048] Positive-negative selection requires that the fourth DNA
sequence encoding the negative marker be substantially incapable of
homologous recombination with the target DNA sequence. In
particular, the fourth DNA sequence should be substantially
non-homologous to a third region of the target DNA. When the fourth
DNA sequence is positioned 3' to the second DNA sequence, the
fourth DNA sequence is non-homologous to a third region of the
target DNA which is adjacent to the second region of the target
DNA. See FIG. 1. When the fourth DNA sequence is located 5' to the
first DNA sequence, it is non-homologous to a fourth region of the
target DNA sequence adjacent to the first region of the target
DNA.
[0049] In some cases, the PNS vector of the invention may be
constructed with a fifth DNA sequence also encoding a negative
selection marker. In such cases, the fifth DNA sequence is
positioned at the opposite end of the PNS vector to that containing
the fourth DNA sequence. The fourth DNA sequence is substantially
non-homologous to the third region of the target DNA and the fifth
DNA sequence is substantially non-homologous to the fourth region
of the target DNA. The negative selection markers contained in such
a PNS vector may either be the same or different negative selection
markers. When they are different such that they require the use of
two different agents to select again cells containing such negative
markers, such negative selection may be carried out sequentially or
simultaneously with appropriate agents for the negative selection
marker. The positioning of two negative selection markers at the 5'
and 3' end of a PNS vector further enhances selection against
target cells which have randomly integrated the PNS vector. This is
because random integration sometimes results in the rearrangement
of the PNS vector resulting in excision of all or part of the
negative selection marker prior to random integration. When this
occurs, cells randomly integrating the PNS vector cannot be
selected against. However, the presence of a second negative
selection marker on the PNS vector substantially enhances the
likelihood that random integration will result in the insertion of
at least one of the two negative selection markers.
[0050] The substantial non-homology between the fourth DNA sequence
(and in some cases fourth and fifth DNA sequences) of the PNS
vector and the target DNA creates a discontinuity in sequence
homology at or near the juncture of the fourth DNA sequence. Thus,
when the vector is integrated into the genome by way of the
homologous recombination mechanism of the cell, the negative
selection marker in the fourth DNA sequence is not transferred into
the target DNA. It is the non-integration of this negative
selection marker during homologous recombination which forms the
basis of the PNS method of the invention.
[0051] As used herein, a "modifying DNA sequence" is a DNA sequence
contained in the first, second and/or third DNA sequence which
encodes the substitution, insertion and/or deletion of one or more
nucleotides in the target DNA sequence after homologous insertion
of the PNS vector into the targeted region of the genome. When the
PNS vector contains only the insertion of the third DNA sequence
encoding the positive selection marker, the third DNA sequence is
sometimes referred to as a "first modifying DNA sequence". When in
addition to the third DNA sequence, the PNS vector also encodes the
further substitution, insertion and/or deletion of one or more
nucleotides, that portion encoding such further modification is
sometimes referred to as a "second modifying DNA sequence". The
second modifying DNA sequence may comprise the entire first and/or
second DNA sequence or in some instances may comprise less than the
entire first and/or second DNA sequence. The latter case typically
arises when, for example, a heterologous gene is incorporated into
a PNS vector which is designed to place that heterologous gene
under the regulatory control of endogenous regulatory sequences. In
such a case, the homologous portion of, for example, the first DNA
sequence may comprise all or part of the targeted endogenous
regulatory sequence and the modifying DNA sequence comprises that
portion of the first DNA sequence (and in some cases a part of the
second DNA sequence as well) which encodes the heterologous DNA
sequence. An appropriate homologous portion in the second DNA
sequence will be included to complete the targeting of the PNS
vector. On the other hand, the entire first and/or second DNA
sequence may comprise a second modifying DNA sequence when, for
example, either or both of these DNA sequences encode for the
correction of a genetic defect in the targeted DNA sequence.
[0052] As used herein, "modified target DNA sequence" refers to a
DNA sequence in the genome of a targeted cell which has been
modified by a PNS vector. Modified DNA sequences contain the
substitution, insertion and/or deletion of one or more nucleotides
in a first transformed target cell as compared to the cells from
which such transformed target cells are derived. In some cases,
modified target DNA sequences are referred to as "first" and/or
"second modified target DNA sequences". These correspond to the DNA
sequence found in the transformed target cell when a PNS vector
containing a first or second modifying sequence is homologously
integrated into the target DNA sequence.
[0053] "Transformed target cells" sometimes referred to as "first
transformed target cells" refers to those target cells wherein the
PNS vector has been homologously integrated into the target cell
genome. A "transformed cell" on the other hand refers to a cell
where in the PNS has non-homologously inserted into the genome
randomly. "Transformed target cells" generally contain a positive
selection marker within the modified target DNA sequence. When the
object of the genomic modification is to disrupt the expression of
a particular gene, the positive selection marker is generally
contained within an exon which effectively disrupts transcription
and/or translation of the targeted endogenous gene. When, however,
the object of the genomic modification is to insert an exogenous
gene or correct an endogenous gene defect, the modified target DNA
sequence in the first transformed target cell will in addition
contain exogenous DNA sequences or endogenous DNA sequences
corresponding to those found in the normal, i.e., nondefective,
endogenous gene.
[0054] "Second transformed target cells" refers to first
transformed target cells whose genome has been subsequently
modified in a predetermined way. For example, the positive
selection marker contained in the genome of a first transformed
target cell can be excised by homologous recombination to produce a
second transformed target cell. The details of such a predetermined
genomic manipulation will be described in more detail
hereinafter.
[0055] As used herein, "heterologous DNA" refers to a DNA sequence
which is different from that sequence comprising the target DNA
sequence. Heterologous DNA differs from target DNA by the
substitution, insertion and/or deletion of one or more nucleotides.
Thus, an endogenous gene sequence may be incorporated into a PNS
vector to target its insertion into a different regulatory region
of the genome of the same organism. The modified DNA sequence so
obtained is a heterologous DNA sequence. Heterologous DNA sequences
also include endogenous sequences which have been modified to
correct or introduce gene defects or to change the amino acid
sequence encoded by the endogenous gene. Further, heterologous DNA
sequences include exogenous DNA sequences which are not related to
endogenous sequences, e.g. sequences derived from a different
species. Such "exogenous DNA sequences" include those which encode
exogenous polypeptides or exogenous regulatory sequences. For
example, exogenous DNA sequences which can be introduced into
murine or bovine ES cells for tissue specific expression (e.g. in
mammary secretory cells) include human blood factors such as t-PA,
Factor VIII, serum albumin and the like. DNA sequences encoding
positive selection markers are further examples of heterologous DNA
sequences.
[0056] The PNS vector is used in the PNS method to select for
transformed target cells containing the positive selection marker
and against those transformed cells containing the negative
selection marker. Such positive-negative selection procedures
substantially enrich for those transformed target cells wherein
homologous recombination has occurred. As used herein, "substantial
enrichment" refers to at least a two-fold enrichment of transformed
target cells as compared to the ratio of homologous transformants
versus nonhomologous transformants, preferably a 10-fold
enrichment, more preferably a 1000-fold enrichment, most preferably
a 10,000-fold enrichment, i.e., the ratio of transformed target
cells to transformed cells. In some instances, the frequency of
homologous recombination versus random integration is of the order
of 1 in 1000 and in some cases as low as 1 in 10,000 transformed
cells. The substantial enrichment obtained by the PNS vectors and
methods of the invention often result in cell populations where in
about 1%, and more preferably about 20%, and most preferably about
95% of the resultant cell population contains transformed target
cells wherein the PNS vector has been homologously integrated. Such
substantially enriched transformed target cell populations may
thereafter be used for subsequent genetic manipulation, for cell
culture experiments or for the production of transgenic organisms
such as transgenic animals or plants.
[0057] FIGS. 2a and 2b show the consequences of gene targeting
(homologous recombination) and random integration of a PNS vector
into the genome of a target cell. The PNS vector shown contains a
neomycin resistance gene as a positive selection marker (neo.sup.r)
and a herpes simplex virus thymidine kinase (HSV-tk) gene as a
negative selection marker. The neo.sup.r positive selection marker
is positioned in an exon of gene X. This positive selection marker
is constructed such that it's expression is under the independent
control of appropriate regulatory sequences. Such regulatory
sequences may be endogenous to the host cell in which case they are
preferably derived from genes actively expressed in the cell type.
Alteratively, such regulatory sequences may be inducible to permit
selective activation of expression of the positive selection
marker.
[0058] On each side of the neo.sup.r marker are DNA sequences
homologous to the regions 5' and 3' from the point of neo.sup.r
insertion in the exon sequence. These flanking homologous sequences
target the X gene for homologous recombination with the PNS vector.
Consistent with the above description of the PNS vector, the
negative selection marker HSV-tk is situated outside one of the
regions of homology. In this example it is 3' to the transcribed
region of gene X. The neo.sup.r gene confers resistance to the drug
G418 (G418.sup.R) whereas the presence of the HSV-tk gene renders
cells containing this gene sensitive to gancyclovir (GANC.sup.s).
When the PNS vector is randomly inserted into the genome by a
mechanism other than by homologous recombination (FIG. 2b),
insertion is most frequently via the ends of the linear DNA and
thus the phenotype for such cells is neo.sup.+ HSV-tk.sup.+
(G418.sup.R, GANC.sup.S). When the PNS vector is incorporated into
the genome by homologous recombination as in FIG. 2a, the resultant
phenotype is neo.sup.+, HSV-tk.sup.- (G418.sup.R, GANC.sup.R).
Thus, those cells wherein random integration of the PNS vector has
occurred can be selected against by treatment with GANC. Those
remaining transformed target cells wherein homologous recombination
has been successful can then be selected on the basis of neomycin
resistance and GANC resistance. It, of course, should be apparent
that the order of selection for and selection against a particular
genotype is not important and that in some instances positive and
negative selection can occur simultaneously.
[0059] As indicated, the neomycin resistance gene in FIG. 2 is
incorporated into an exon of gene X. As so constructed, the
integration of the PNS vector by way of homologous recombination
effectively blocks the expression of gene X. In multicellular
organisms, however, integration is predominantly random and occurs,
for the most part, outside of the region of the genome encoding
gene X. Non-homologous recombination therefore will not disrupt
gene X in most instances. The resultant phenotypes will therefore,
in addition to the foregoing, will also be X.sup.- for homologous
recombination and X.sup.+ for random integration. In many cases it
is desirable to disrupt genes by positioning the positive selection
marker in an exon of a gene to be disrupted or modified. For
example, specific proto-oncogenes can be mutated by this method to
produce transgenic animals. Such transgenic animals containing
selectively inactivate proto-oncogenes are useful in dissecting the
genetic contribution of such a gene to oncogenesis and in some
cases normal development.
[0060] Another potential use for gene inactivation is disruption of
proteinaceous receptors on cell surfaces. For example, cell lines
or organisms wherein the expression of a putative viral-receptor
has been disrupted using an appropriate PNS vector can be assayed
with virus to confirm that the receptor is, in fact, involved in
viral infection. Further, appropriate PNS vectors may be used to
produce transgenic animal models for specific genetic defects. For
example, many gene defects have been characterized by the failure
of specific genes to express functional gene product, e.g. .alpha.
and .beta. thalassema, hemophilia, Gaucher's disease and defects
affecting the production of .alpha.-1-antitrypsin, ADA, PNP,
phenylketonurea, familial hypercholesterolemia and retinoblastemia.
Transgenic animals containing disruption of one or both alleles
associated with such disease states or modification to encode the
specific gene defect can be used as models for therapy. For those
animals which are viable at birth, experimental therapy can be
applied. When, however, the gene defect affects survival, an
appropriate generation (e.g. F0, F1) of transgenic animal may be
used to study in vivo techniques for gene therapy.
[0061] A modification of the foregoing means to disrupt gene X by
way of homologous integration involves the use of a positive
selection marker which is deficient in one or more regulatory
sequences necessary for expression. The PNS vector is constructed
so that part but not all of the regulatory sequences for gene X are
contained in the PNS vector 5' from the structural gene segment
encoding the positive selection marker, e.g., homologous sequences
encoding part of the promotor of the X gene. As a consequence of
this construction, the positive selection marker is not functional
in the target cell until such time as it is homologously integrated
into the promotor region of gene X. When so integrated, gene X is
disrupted and such cells may be selected by way of the positive
selection marker expressed under the control of the target gene
promoter. The only limitation in using such an approach is the
requirement that the targeted gene be actively expressed in the
cell type used. Otherwise, the positive selection marker will not
be expressed to confer a positive selection characteristic on the
cell.
[0062] In many instances, the disruption of an endogenous gene is
undesirable, e.g., for some gene therapy applications. In such
situations, the positive selection marker comprising the third DNA
sequence of the PNS vector may be positioned within an untranslated
sequence, e.g. an intron of the target DNA or 5' or 3' untranslated
regions. FIG. 3 depicts such a PNS vector. As indicated, the first
DNA sequence comprises part of exon I and a portion of a contiguous
intron in the target DNA. The second DNA sequence encodes an
adjacent portion of the same intron and optionally may include all
or a portion of exon II. The positive selection marker of the third
DNA sequence is positioned between the first and second sequences.
The fourth DNA sequence encoding the negative selection marker, of
course, is positioned outside of the region of homology. When the
PNS vector is integrated into the target DNA by way of homologous
recombination the positive selection marker is located in the
intron of the targeted gene. The third DNA sequence is constructed
such that it is capable of being expressed and translated
independently of the targeted gene. Thus, it contains an
independent functional promotor, translation initiation sequence,
translation termination sequence, and in some cases a
polyadenylation sequence and/or one or more enhancer sequences,
each functional in the cell type transfected with the PNS vector.
In this manner, cells incorporating the PNS vector by way of
homologous recombination can be selected by way of the positive
selection marker without disruption of the endogenous gene. Of
course, the same regulatory sequences can be used to control the
expression of the positive selection marker when it is positioned
within an exon. Further, such regulatory sequences can be used to
control expression of the negative selection marker. Regulatory
sequences useful in controlling the expression of positive and/or
negative selection markers are listed in Table IIB. Of course,
other regulatory sequences may be used which are known to those
skilled in the art. In each case, the regulatory sequences will be
properly aligned and, if necessary, placed in proper reading frame
with the particular DNA sequence to be expressed. Regulatory
sequence, e.g. enhancers and promoters from different sources may
be combined to provide modulated gene expression.
2TABLE IIA Tissue Specific Regulatory Sequences Cell/ Promoter/
Tissue Enhancer Reference Adrenal PNMT Baetge, et al. (1988) PNAS
85 Erythoroid .beta.-globin Townes et al. (1985) EMBO J 4: 1715
Lens .alpha.-crystallin Overteek et al. (1985) PNAS 82: 7815 Liver
.alpha.-FP Krumlauf et al. (1985) MCB 5: 1639 Lymphoid Ig.mu.
(.gamma. - 1) Yamamura et al. (1986) promoter/ PNAS 83: 2152
enhancer Mammary WAP Gordon et al. (1987) Bio/Tech 5: 1183 Nervous
MBP Tamura et al. (1989) MCB 9: 3122 Pancreas (B) Insulin Hanaban
(1985) Nature 315: 115 Pancreas Elastase Swift et al. (1984)
(exocrine) Cell 38: 639 Pituitary Prolactin Ingraham et al. (1988)
Cell 55: 579 Skeletal ckm Johnson et al. (1989) Muscle MCB 9: 3393
Testes Protamine Stewart et al. (1988) MCB 8: 1748
[0063]
3TABLE IIB Regulatory Sequences for Use With Positive and/or
Negative Selection Markers Regulatory Sequence Cell Type PYF441
enhancer/HSV-tk promoter embryo-derived (pMCI-Neo control) ASV-LTR
fibroblasts SV-40 early variety of mammalian cells Cytomegalo virus
general mammalian .beta.-actin general mammalian MoMuLV haemopoetic
stem cells SFFV haemopoetic stem cells Mannopine synthase general
plant Octapine synthase general plant Nopaline synthase general
plant Cauliflower mosiac virus 35S general plant promoter/enhancer
.beta.-phaseolin seeds "insert-7" protoplasts
[0064] A modification of the target DNA sequence is also shown in
FIG. 3. In exon I of the target DNA sequence, the sixth codon GTG
is shown which encodes valine. In the first DNA sequence of the PNS
vector, the codon GAG replaces the GTG codon in exon I. This latter
codon encodes glutamine. Cells selected for homologous
recombination as a consequence encode a modified protein wherein
the amino acid encoded by the sixth codon is changed from valine to
glutamine.
[0065] There are, of course, numerous other examples of
modifications of target DNA sequences in the genome of the cell
which can be obtained by the PNS vectors and methods of the
invention. For example, endogenous regulatory sequences controlling
the expression of proto-oncogenes can be replaced with regulatory
sequences such as promoters and/or enhancers which actively express
a particular gene in a specific cell type in an organism, i.e.,
tissue-specific regulatory sequences. In this manner, the
expression of a proto-oncogene in a particular cell type, for
example in a transgenic animal, can be controlled to determine the
effect of oncogene expression in a cell type which does not
normally express the proto-oncogene. Alternatively, known viral
oncogenes can be inserted into specific sites of the target genome
to bring about tissue-specific expression of the viral oncogene.
Examples of preferred tissue-specific regulatory sequences are
listed in Table IIA. Examples of proto-oncogenes which may be
modified by the PNS vectors and methods to produce tissue specific
expression and viral oncogenes which may be placed under control of
endogenous regulatory sequences are listed in Table IIIA and IIIB,
respectively.
4TABLE IIIA Proto-oncogenes involved in human tumors Gene Disease
c-abl chronic myelogenous leukemia c-erbB squamous cell carcinoma
glial blastoma c-myc Burkitt's lymphoma small cell carcinoma of
lung carcinoma of breat L-myc small cell carcinoma of lung N-myc
small cell carcinoma of lung neuroblastoma neu carcinoma of breast
C-ras variety
[0066]
5TABLE IIIB Viral oncogenes known to cause tumors when ectopically
expressed in mice Ha-ras Sv40Tag HPV-E6 v-abl HPV-E7 v-fps PyTag
v-myc v-src
[0067] As indicated, the positive-negative selection methods and
vectors of the invention are used to modify target DNA sequences in
the genome of target cells capable of homologous recombination.
Accordingly, the invention may be practiced with any cell type
which is capable of homologous recombination. Examples of such
target cells include cells derived from vertebrates including
mammals such as humans, bovine species, ovine species, murine
species, simian species, and other eucaryotic organisms such as
filamentous fungi, and higher multicellular organisms such as
plants. The invention may also be practiced with lower organisms
such as gram positive and gram negative bacteria capable of
homologous recombination. However, such lower organisms are not
preferred because they generally do not demonstrate significant
non-homologous recombination, i.e., random integration.
Accordingly, there is little or no need to select against
non-homologous transformants.
[0068] In those cases where the ultimate goal is the production of
a non-human transgenic animal, embryonic stem cells (ES cells) are
preferred target cells. Such cells have been manipulated to
introduce transgenes. ES cells are obtained from pre-implantation
embryos cultured in vitro. Evans, M. J., et al. (1981), Nature,
292, 154-156; Bradley, M. O., et al. (1984), Nature, 309, 255-258;
Gossler, et al. (1986), Proc. Natl. Acad. Sci. USA, 83, 9065-9069;
and Robertson, et al. (1986), Nature, 322, 445-448. PNS vectors can
be efficiently introduced into the ES cells by electroporation or
microinjection or other transformation methods, preferably
electroporation. Such transformed ES cells can thereafter be
combined with blastocysts from a non-human animal. The ES cells
thereafter colonize the embryo and can contribute to the germ line
of he resulting chimeric animal. For review see Jaenisch, R.
(1988), Science, 240, 1468-1474. In the present invention, PNS
vectors are targeted to a specific portion of the ES cell genome
and thereafter used to generate chimeric transgenic animals by
standard techniques.
[0069] When the ultimate goal is gene therapy to correct a genetic
defect in an organism such as a human being, the cell type will be
determined by the etiology of the particular disease and how it is
manifested. For example, hemopoietic stem cells are a preferred
cells for correcting genetic defects in cell types which
differentiate from such stem cells, e.g. erythrocytes and
leukocytes. Thus, genetic defects in globin chain synthesis in
erythrocytes such as sickle cell anemia, .beta.-thalassemia and the
like may be corrected by using the PNS vectors and methods of the
invention with hematopoietic stem cells isolated from an affected
patient. For example, if the target DNA in FIG. 3 is the
sickle-cell .beta.-globin gene contained in a hematopoietic stem
cell and the PNS vector in FIG. 3 is targeted for this gene with
the modification shown in the sixth codon, transformed
hematopoietic stem cells can be obtained wherein a normal
.beta.-globin will be expressed upon differentiation. After
correction of the defect, the hematopoietic stem cells may be
returned to the bone marrow or systemic circulation of the patient
to form a subpopulation of erythrocytes containing normal
hemoglobin. Alternatively, hematopoietic stem cells may be
destroyed in the patient by way of irradiation and/or chemotherapy
prior to reintroduction of the modified hematopoietic stem cell
thereby completely rectifying the defect.
[0070] Other types of stem cells may be used to correct the
specific gene defects associated with cells derived from such stem
cells. Such other stem cells include epithelial, liver, lung,
muscle, endothelial, menchymal, neural and bone stem cells. Table
IV identifies a number of known genetic defects which ar amenable
to correction by the PNS methods and vectors of the invention.
[0071] Alternatively, certain disease states can be treated by
modifying the genome of cells in a way which does not correct a
genetic defect per se but provides for the supplementation of the
gene product of a defective gene. For example, endothelial cells
are preferred as targets for human gene therapy to treat disorders
affecting factors normally present in the systemic circulation. In
model studies using both dogs and pigs endothelial cells have been
shown to form primary cultures, to be transformable with DNA in
culture, and to be capable of expressing a transgene upon
re-implantation in arterial grafts into the host organism. Wilson,
et al. (1989), Science, 244, 1344; Nabel, et al. (1989), Science,
244, 1342. Since endothelial cells form an integral part of the
graft, such transformed cells can be used to produce proteins to be
secreted into the circulatory system and thus serve as therapeutic
agents in the treatment of genetic disorders affecting circulating
factors. Examples of such diseases include insulin-deficient
diabetes, .alpha.-1-antitrypsin deficiency, and hemophilia.
Epithelial cells provide a particular advantage in the treatment of
factor VIII-deficient hemophilia. These cells naturally produce von
Willebrand factor and it has been shown that production of active
factor VIII is dependant upon the autonomous synthesis of vWF
(Toole, et al. (1986), Proc. Natl. Acad. Sci. USA, 83, 5939).
[0072] As indicated in Example 4, human endothelial cells from a
hemophiliac patient deficient in Factor VIII are modified by a PNS
vector to produce an enriched population of transformed endothelial
cells wherein the expression of DNA sequences encoding a secretory
form of Factor VIII is placed under the control of the regulatory
sequences of the endogenous .beta.-actin gene. Such transformed
cells are implanted into vascular grafts from the patient. After
incorporation of transformed cells, it is grafted back into the
vascular system of the patient. The transformed cells secrete
Factor XIII into the vascular system to supplement the defect in
the patients blood clotting system.
[0073] Other diseases of the immune and/or the circulatory system
are candidates for human gene therapy. The target tissue, bone
marrow, is readily accessible by current technology, and advances
are being made in culturing stem cells in vitro. The immune
deficiency diseases caused by mutations in the enzymes adenosine
deaminase (ADA) and purine nucleotide phosphorylase (PNP), are of
particular interest. Not only have the genes been cloned, but cells
corrected by PNS gene therapy are likely to have a selective
advantage over their mutant counterparts. Thus, ablation of the
bone marrow in recipient patients may not be necessary.
[0074] The PNS approach is applicable to genetic disorders with the
following characteristics: first, the DNA sequence and preferably
the cloned normal gene must be available; second, the appropriate,
tissue relevant, stem cell or other appropriate cell must be
available. Below is Table IV listing some of the known genetic
diseases, the name of the cloned gene, and the tissue type in which
therapy may be appropriate. These and other genetic disease
amenable to the PNS methods and vectors of the invention have been
reviewed. See Friedman (1989), Science, 244, 1275; Nichols, E. K.
(1988), Human Gene Therapy (Harvard University Press); and Cold
Springs Harbor Symposium on Quantitative Biology, V 1. 11 (1986),
"The Biology of Homo Sapiens" (Cold Springs Harbor Press).
6TABLE IV Human Genetic Diseases in Which the Disease Locus has
been Cloned Target Disease Gene Tissue .alpha.1-anti-trypsin
.alpha.1-anti trypsin liver disease Gaucher Disease
glucocerebrosidase bone marrow Granulocyte Actin Granulocyte Actin
bone marrow Deficiency Immunodeficiency Adenosine deaminase bone
marrow Immunodeficiency Purine nucleoside bone marrow Muscular most
likely skeletal Dystrophy dystropin gene muscle Phenylketonuria
Phenylalanine liver hydroxylase Sickle Cell .beta.-globin bone
marrow Anemia Thalassemia globin bone marrow Hemophilia various
clotting bone marrow/ factors endothelial cells Familial hyper- low
density liver/endo- cholesterolemia lipoprotein endothelial
receptor cells
[0075] As indicated, genetic defects may be corrected in specific
cell lines by positioning the positive selection marker (the second
DNA sequence in the PNS vector) in an untranslated region such as
an intron near the site of the genetic defect together with
flanking segments to correct the defect. In this approach, the
positive selection marker is under its own regulatory control and
is capable of expressing itself without substantially interfering
with the expression of the targeted gene. In the case of human gene
therapy, it may be desirable to introduce only those DNA sequences
which are necessary to correct the particular genetic defect. In
this regard, it is desirable, although not necessary, to remove the
residual positive selection marker which remains after correction
of the genetic defect by homologous recombination.
[0076] The removal of a positive selection marker from a genome in
which homologous insertion of a PNS vector has occurred can be
accomplished in many ways. For example, the PNS vector can include
a second negative selection marker contained within the second DNA
sequence. This second negative selection marker is different from
the first negative selection marker contained in the fourth DNA
sequence. After homologous integration, a second modified target
DNA sequence is formed containing the third DNA encoding both the
positive selection marker and the second negative selection marker.
After isolation and purification of the first transformed target
cells by way of negative selection against transformed cells
containing the first negative selection marker and for those cells
containing the positive selection marker, the first transformed
target cells are subjected to a second cycle of homologous
recombination. In this second cycle, a second homologous vector is
used which contains all or part of the first and second DNA
sequence of the PNS vector (encoding the second modification in the
target DNA) but not those sequences encoding the positive and
second negative selection markers. The second negative selection
marker in the first transformed target cells is then used to select
against unsuccessful transformants and cells where in the second
homologous vector is randomly integrated into the genome.
Homologous recombination of this second homologous vector, however,
with the second modified target DNA sequence results in a second
transformed target cell type which does not contain either the
positive selection marker or the second negative selection marker
but which retains the modification encoded by the first and/or
second DNA sequences. Cells which have not homologously integrated
the second homologous vector are selected against using the second
negative selection marker.
[0077] The PNS vectors and methods of the invention are also
applicable to the manipulation of plant cells and ultimately the
genome of the entire plant. A wide variety of transgenic plants
have been reported, including herbaceous dicots, woody dicots and
monocots. For a summary, see Gasser, et al. (1989), Science, 244,
1293-1299. A number of different gene transfer techniques have been
developed for producing such transgenic plants and transformed
plant cells. One technique used Agrobacterium tumefaciens as a gene
transfer system. Rogers, et al. (1986), Methods Enzymol., 118,
627-640. A closely related transformation utilizes the bacterium
Agrobacterium rhizogenes. In each of these systems a Ti or Ri plant
transformation vector can be constructed containing border regions
which define the DNA sequence to be inserted into the plant genome.
These systems previously have been used to randomly integrate
exogenous DNA to plant genomes. In the present invention, an
appropriate PNS vector may be inserted into the plant
transformation vector between the border sequences defining the DNA
sequences transferred into the plant cell by the Agrobacterium
transformation vector.
[0078] Preferably, the PNS vector of the invention is directly
transferred to plant protoplasts by way of methods analogous to
that previously used to introduce transgenes into protoplasts. See,
e.g. Paszkowski, et al. (1984), EMBO J., 3, 2717-2722; Hain, et al.
(1985), Mol. Gen. Genet., 199, 161-168; Shillito, et al. (1985),
Bio/Technology, 3, 1099-1103; and Negrutiu, et al. (1987), Plant
Mol. Bio., 8, 363-373. Alternatively, the PNS vector is contained
within a liposome which may be fused to a plant protoplast (see,
e.g. Deshayes, et al. (1985), EMBO J., 4, 2731-2738) or is directly
inserted to plant protoplast by way of intranuclear microinjection
(see, e.g. Crossway. et al. (1986), Mol. Gen Genet., 202, 179-185,
and Reich, et al. (1986), Bio/Technology, 4, 1001-1004).
Microinjection is the preferred method for transfecting
protoplasts. PNS vectors may also be microinjected into
meristematic inflorenscences. De la Pena et al. (1987), Nature,
325, 274-276. Finally, tissue explants can be transfected by way of
a high velocity microprojectile coated with the PNS vector
analogous to the methods used for insertion of transgenes. See,
e.g. Vasil (1988), Bio/Technology, .sctn., 397; Klein, et al.
(9187), Nature, 327, 70; Klein, et al. (1988), Proc. Natl. Acad.
Sci. USA, 85, 8502; McCabe, et al. (1988), Bio/Technology, 6, 923;
and Klein, et al., Genetic Engineering, Vol 11, J. K. Setlow editor
(Academic Press, N.Y., 1989). Such transformed explants can be used
to regenerate for example various serial crops. Vasil (1988),
Bio/Technology, 6, 397.
[0079] Once the PNS vector has been inserted into the plant cell by
any of the for going methods, homologous or combination targets the
PNS vector to the appropriate site in the plant genome. Depending
upon the methodology used to transfect, positive-negative selection
is performed on tissue cultures of the transformed protoplast or
plant cell. In some instances, cells amenable to tissue culture may
be excised from a transformed plant either from the F0 or a
subsequent generation.
[0080] The PNS vectors and method of the invention are used to
precisely modify the plant genome in a predetermined way. Thus, for
example, herbicide, insect and disease resistance may be
predictably engineered into a specific plant species to provide,
for example, tissue specific resistance, e.g., insect resistance in
leaf and bark. Alternatively, the expression levels of various
components within a plant may be modified by substituting
appropriate regulatory elements to change the fatty acid and/or oil
content in seed, the starch content within the plant and the
elimination of components contributing to undesirable flavors in
food. Alternatively, heterologous genes may be introduced into
plants under the predetermined regulatory control in the plant to
produce various hydrocarbons including waxes and hydrocarbons used
in the production of rubber.
[0081] The amino acid composition of various storage proteins in
wheat and corn, for example, which are known to be deficient in
lysine and tryptophan may also be modified. PNS vectors can be
readily designed to alter specific codons within such storage
proteins to encode lysine and/or tryptophan thereby increasing the
nutritional value of such crops. For example, the zein protein in
corn (Pederson et al. (1982), Cell, 29, 1015) may be modified to
have a higher content of lysine and tryptophan by the vectors and
methods of the invention.
[0082] It is also possible to modify the levels of expression of
various positive and negative regulatory elements controlling the
expression of particular proteins in various cells and organisms.
Thus, the expression level of negative regulatory elements may be
decreased by use of an appropriate promotor to enhance the
expression of a particular protein or proteins under control of
such a negative regulatory element. Alternatively, the expression
level of a positive regulatory protein may be increased to enhance
expression of the regulated protein or decreased to reduce the
amount of regulated protein in the cell or organism.
[0083] The basic elements of the PNS vectors of the invention have
already been described. The selection of each of the DNA sequences
comprising the PNS vector, however, will depend upon the cell type
used, the target DNA sequence to be modified and the type of
modification which is desired.
[0084] Preferably, the PNS vector is a linear double stranded DNA
sequence. However, circular closed PNS vectors may also be used.
Linear vectors are preferred since they enhance the frequency of
homologous integration into the target DNA sequence. Thomas, et al.
(1986), Cell, 44, 49.
[0085] In general, the PNS vector (including first, second, third
and fourth DNA sequences) has a total length of between 2.5 kb
(2500 base pairs) and 1000 kb. The lower size limit is set by two
criteria. The first of these is the minimum necessary length of
homology between the first and second sequences of the PNS vector
and the target locus. This minimum is approximately 500 bp (DNA
sequence 1 plus DNA sequence 2). The second criterion is the need
for functional genes in the third and fourth DNA sequences of the
PNS vector. For practical reasons, this lower limit is
approximately 1000 bp for each sequence. This is because the
smallest DNA sequences encoding known positive and negative
selection markers are about 1.0-1.5 kb in length.
[0086] The upper limit to the length of the PNS vector is
determined by the state of the technology used to manipulate DNA
fragments. If these fragments are propagated as bacterial plasmids,
a practical upper length limit is about 25 kb; if propagated as
cosmids, the limit is about 50 kb, if propagated as YACs (yeast
artificial chromosomes) the limit approaches 1000 kb (Burke, et al.
(1987), Science, 236, 806).
[0087] Within the first and second DNA sequences of the PNS vector
are portions of DNA sequence which are substantially homologous
with sequence portions contained within the first and second
regions of the target DNA sequence. The degree of homology between
the vector and target sequences influences the frequency of
homologous recombination between the two sequences. One hundred
percent sequence homology is most preferred, however, lower
sequence homology can be used to practice the invention. Thus,
sequence homology as low as about 80% can be used. A practical
lower limit to sequence homology can be defined functionally as
that amount of homology which if further reduced does not mediate
homologous integration of the PNS vector into the genome. Although
as few as 25 bp of 100% homology are required for homologous
recombination in mammalian cells (Ayares, et al. (1986), Genetics,
83, 5199-5203), longer regions are preferred, e.g., 500 bp, more
preferably, 5000 bp, and most preferably, 25000 bp for each
homologous portion. These numbers define the limits of the
individual lengths of the first and second sequences. Preferably,
the homologous portions of the PNS vector will be 100% homologous
to the target DNA sequence, as increasing the amount of
non-homology will result in a corresponding decrease in the
frequency of gene targeting. If non-homology does exist between the
homologous portion of the PNS vector and the appropriate region of
the target DNA, it is preferred that the non-homology not be spread
throughout the homologous portion but rather in discrete areas of
the homologous portion. It is also preferred that the homologous
portion of the PNS vector adjacent to the negative selection marker
(fourth or fifth DNA sequence) be 100% homologous to the
corresponding region in the target DNA. This is to ensure maximum
discontinuity between homologous and non-homologous sequences in
the PNS vector.
[0088] Increased frequencies of homologous recombination have been
observed when the absolute amount of DNA sequence in the combined
homologous portions of the first and second DNA sequence are
increased. FIG. 4 depicts the targeting frequency of the Hprt locus
as a function of the extent of homology between an appropriate PNS
vector and the endogenous target. A series of replacement
(.tangle-solidup.) and insertion (.circle-solid.) Hprt vectors were
constructed that varied in the extent of homology to the endogenous
Hprt gene. Hprt sequences in each vector were interrupted in the
eighth exon with the neomycin resistance gene. The amount of Hprt
sequence 3' to the neo gene was kept constant to the amount of Hprt
sequence 5' to the neo was varied. The absolute frequency of
independent targeting events per total ES cells electroporated is
plotted in FIG. 4 on the logarithmic scale as a function of the
number of kilobases of Hprt sequence contained within the PNS
vectors. See Capacchi, M. R. (1989), Science, 244, 1288-1292.
[0089] As previously indicated, the fourth DNA sequence containing
the negative selection marker should have sufficient non-homology
to the target DNA sequence to prevent homologous recombination
between the fourth DNA sequence and the target DNA. This is
generally not a problem since it is unlikely that the negative
selection marker chosen will have any substantial homology to the
target DNA sequence. In any event, the sequence homology between
the fourth DNA sequence and the target DNA sequence should be less
than about 50%, most preferably less than about 30%.
[0090] A preliminary assay for sufficient sequence non-homology
between the fourth DNA sequence and the target DNA sequence
utilizes standard hybridization techniques. For example, the
particular negative selection marker may be appropriately labeled
with a radioisotope or other detectable marker and used as a probe
in a Southern blot analysis of the genomic DNA of the target cell.
If little or no signal is detected under intermediate stringency
conditions such as 3.times.SSC when hybridized at about 55.degree.
C., that negative selection marker should be functional in a PNS
vector designed for homologous recombination in that cell type.
However, even if a signal is detected, it is not necessarily
indicative that particular negative selection cannot be used in a
PNS vector targeted for that genome. This is because the negative
selection marker may be hybridizing with a region of the genome
which is not in proximity with the target DNA sequence. Since the
target DNA sequence is defined as those DNA sequences corresponding
to first, second, third, and in some cases, fourth regions of the
genome, Southern blots localizing the regions of the target DNA
sequence may be performed. If the probe corresponding to the
particular negative selection marker does not hybridize to these
bands, it should be functional for PNS vectors directed to these
regions of the genome.
[0091] Hybridization between sequences encoding the negative
selection marker and the genome or target regions of a genome,
however, does not necessarily mean that such a negative selection
marker will not function in a PNS vector. The hybridization assay
is designed to detect those sequences which should function in the
PNS vector because of their failure to hybridize to the target.
Ultimately, a DNA sequence encoding a negative selection marker is
functional in a PNS vector if it is not integrated during
homologous recombination regardless of whether or not it hybridizes
with the target DNA.
[0092] It is also possible that high stringency hybridization can
be used to ascertain whether genes from one species can be targeted
into related genes in a different species. For example, preliminary
gene therapy experiments may require that human genomic sequences
replace the corresponding related genomic sequence in mouse cells.
High stringency hybridization conditions such as 0.1.times.SSC at
about 68.degree. C. can be used to correlate hybridization signal
under such conditions with the ability of such sequences to act as
homologous portions in the first and second DNA sequence of the PNS
vector. Such experiments can be routinely performed with various
genomic sequences having known differences in homology. The measure
of hybridization may therefor correlate with the ability of such
sequences to bring about acceptable frequencies of
recombination.
[0093] Table I identifies various positive and negative selection
markers which may be used respectively in the third and fourth DNA
sequences of the PNS vector together with the conditions used to
select for or against cells expressing each of the selection
markers. As for animal cells such as mouse L cells, ES cells,
preferred positive selection markers include DNA sequences encoding
neomycin resistance and hygromycin resistance, most preferably
neomycin resistance. For plant cells preferred positive selection
markers include neomycin resistance and bleomycin resistance, most
preferably neomycin resistance.
[0094] For animal cells, preferred negative selection markers
include gpt and HSV-tk, most preferably HSV-tk. For plant cells,
preferred negative selection markers include Gpt and HSV-tk. As
genes responsible for bacterial and fungal pathogenesis in plants
are cloned, other negative markers will become readily
available.
[0095] As used herein, a "positive screening marker" refers to a
DNA sequence used in a phage rescue screening method to detect
homologous recombination. An example of such a positive screening
marker is the supF gene which encodes a tyrosine transfer RNA which
is capable of suppressing amber mutations. See Smithies, et al.
(1985), Nature, 317, 230-234.
[0096] The following is presented by way of example and is not to
be construed as a limitation on the scope of the invention.
EXAMPLE 1
Inactivation at the Int-2 Locus in Mouse ES Cells
[0097] 1. PNS Vector Construction
[0098] The PNS vector, pINT-2-N/TK, is described in Mansour, et al.
(1988), Nature, 336, 349. This vector was used to disrupt the
proto-oncogene, INT-2, in mouse ES cells. As shown in FIG. 5c, it
contains DNA sequences 1 and 2 homologous to the target INT-2
genomic sequences in mouse ES cells. These homologous sequences
were obtained from a plasmid referred to as pAT-153 (Peters, et al.
(1983), Cell, 33, 369). DNA sequence 3, the positive selection
moiety of the PNS vector was the Neo gene from the plasmid pMCINeo
described in Thomas, et al. (1987), Cell, 51, 503; DNA sequence 4,
the negative selection element of the vector, was the HSV-TK gene
derived from the plasmid pIC-19-R/TK which is widely available in
the scientific community.
[0099] Plasmid pIC19R/MC1-TK (FIG. 5d) contains the HSV-TK gene
engineered for expression in ES cells (Mansour, et al. (1988),
Nature, 336, 348-352). The TK gene, flanked by a duplication of a
mutant polyoma virus enhancer, PYF441, has been inserted into the
vector, pIC19R (Marsh, et al. (1984), Gene, 32, 481-485) between
the XhoI and the HindIII sites. The map of plasmid pIC19R/MC1-TK is
shown in FIG. 5d. The enhancer sequence is as follows:
7 5' CTCGAGCAGT GTGGTTTTCA AGAGGAAGCA AAAAGCCTCT CCACCCAGGC
CTGGAATGTT TCCACCCAAT GTOGAGCAGT GTGGTTTTGC AAGAGGAAGC AAAAAGCCTC
TCCACCCAGG CCTGGAATGT TTCCACCCAA TGTCGAG 3'
[0100] The 5' end is an XhoI restriction enzyme site, the 3' end is
contiguous with the HSV-TK gene. The HSV-TK sequences are from
nucleotides 92-1799 (McKnight (1980), Nucl. Acids. Res., 8,
5949-5964) followed at the 3' end by a HindIII linker. The plasmid
pIC19R is essentially identical to the pUC vectors, with an
alternative poly-linker as shown in FIG. 5d.
[0101] Construction of the vector, pINT-2-N/TK involved five
sequential steps as depicted in FIG. 5. First, a 3,965 bp PstI
fragment containing exon 1b, was excised from pAT153 and inserted
into the PstI site of Bluescribe.RTM. (Stratagene of LaJolla,
Calif.), an Amp.sup.R bacterial plasmid containing a multi-enzyme,
cloning polylinker. Second, a synthetic XhoI linker of sequence
1
[0102] was inserted into the ApaI-site on exon 1b. Third, the
XhoI-SalI Neo.sup.r-fragment from pMCI Neo was inserted into the
XhoI linker in exon 1b. Fourth, the 3,965 bp INT-2 Pst fragment
containing the Neo.sup.r gene was reinserted into pAT153, to
generate the plasmid pINT-2-N as shown in FIG. 5b. This plasmid
also includes the third exon of the int-2 gene. Fifth, the
ClaI-HindIII HSV-tk fragment from pIC-19-R/TK was inserted into
ClaI-HindIII digested pINT2-N, creating the final product,
pINT2-N/TK. This vector was linearized by digestion with ClaI prior
to its introduction into ES cells.
[0103] 2. Generation of ES Cells
[0104] ES cells were derived from two sources. The first source was
isolation directly from C57B1/6 blastocysts (Evans, et al. (1981),
Nature, =, 154-156) except that primary embryonic fibroblasts
(Doetschman, et al. (1985), J. Embryol. Exp. Morphol., 87, 27-45)
were used as feeders rather than STO cells. Briefly, 2.5 days
postpregnancy mice were ovariectomized, and delayed blastocysts
were recovered 4-6 days later. The blastocysts were cultured on
mitomycin C-inactivated primary embryonic fibroblasts. After
blastocyst attachment and the outgrowth of the trophectoderm, the
ICM-derived clump was picked and dispersed by trypsin into clumps
of 3-4 cells and put onto new feeders. All culturing was carried
out in DMEM plus 20% FCS and 10.sup.-4 M .beta.-mercaptoethanol.
The cultures were examined daily. After 6-7 days in culture,
colonies that still resembled ES cells were picked, dispersed into
single cells, and replated on feeders. Those cell lines that
retained the morphology and growth characteristic of ES cells were
tested for pluripotency in vitro. These cell lines were maintained
on feeders and transferred every 2-3 days.
[0105] The second method was to utilize one of a number of ES cell
lines isolated from other laboratories, e.g., CC1.2 described by
Kuehn, et al. (1987), Nature, 326, 295. The cells were grown on
mitomycin C-inactivated STO cells. Cells from both sources behaved
identically in gene targeting experiments.
[0106] 3. Introduction of PNS Vector pINT-2-N/TK into ES Cells
[0107] The PNS vector pINT-2-N/TK was introduced into ES cells by
electroporation using the Promega Biotech X-Cell 2000. Rapidly
growing cells were trypsinized, washed in DMEM, counted and
resuspended in buffer containing 20 mM HEPES (pH 7.0), 137 mM NaCl,
5 mM KCl, 0.7 mM Na.sub.2HPO.sub.4, 6 mM dextrose, and 0.1 mM
.beta.-mercaptoethanol. Just prior to electroporation, the
linearized recombinant vector was added. Approximately 25 .mu.g of
linearized PNS vector was mixed with 10.sup.7 ES cells in each 1
ml-cuvette.
[0108] Cells and DNA were exposed to two sequential 625V/cm pulses
at room temperature, allowed to remain in the buffer for 10
minutes, then plated in non-selective media onto feeder cells.
[0109] 4. Selection of ES Cells Containing a Targeted Disruption of
the Int-2 Locus
[0110] Following two days of non-selective growth, the cells were
trypsinized and replated onto G418 (250 .mu.g/ml) media. The
positive-selection was applied alone for three days, at which time
the cells were again trypsinized and replated in the presence of
G418 and either gancyclovir (2.times.10.sup.-6M) (Syntex, Palo
Alto, Calif.) or
1-(2-deoxy-2-fluoro-.beta.-D-arabino-furanosyl-5-iodouracil
(F.I.A.U.) (1.times.10.sup.-6M) (Bristol Myers). When the cells had
grown to confluency, each plate of cells was divided into two
aliquots, one of which was frozen in liquid N.sub.2, the other
harvested for DNA analysis.
[0111] 5. Formation of INT-2 Disrupted Transgenic Mice
[0112] Those transformed cells determined to be appropriately
modified by the PNS vector were grown in non-selective media for
2-5 days prior to injection into blastocysts according to the
method of Bradley in Teratocarcinomas and embryonic stem cells, a
practical approach, edited by E. J. Robertson, IRL Press, Oxford
(1987), p. 125.
[0113] Blastocysts containing the targeted ES cells were implanted
into pseudo-pregnant females and allowed to develop to term.
Chimaeric offspring were identified by coat-color markers and those
males showing chimaerism were selected for breeding offspring.
Those offspring which carry the mutant allele can be identified by
coat color, and the presence of the mutant allele reaffirmed by DNA
analysis by tail-blot, DNA analysis.
EXAMPLE 2
Disruption at the hox1.4 Locus in Mouse ES Cells
[0114] Disruption of the hox1.4 locus was performed by methods
similar to those described to disrupt the int-2 locus. There were
two major differences between these two disruption strategies.
First, the PNS vector, pHOX1.4N/TK-TK2 (FIG. 6), used to disrupt
the hox1.4 locus contained two negative selection markers, i.e., a
DNA sequence 5 encoding a second negative selection marker was
included on the PNS vector at the end opposite to DNA sequence 4
encoding the first negative selection marker. DNA sequence 5
contained the tk gene isolate d from HSV-type 2. It functioned as a
negative-selectable marker by the same method as the original
HSV-tk gene, but the two tk genes are 20% non-homologous. This
non-homology further inhibits r combination between DNA sequences 4
and 5 in the vector which might have inhibited gene-targeting. The
second difference between the int-2 and the hox1.4 disruption
strategies is that the vector pHOX1.4N/TK-TK2 contains a deletion
of 1000 bp of hox1.4 sequences internal to the gene, i.e., DNA
sequences 1 and 2 are not contiguous.
[0115] The HSV-tk2 sequences used in this construction were
obtained from pDG504 (Swain, M. A. et al. (1983), J. Virol., 46,
1045). The structural TK gene from pDG504 was inserted adjacent to
the same promoter/enhancer sequences used to express both the Neo
and HSV-tk genes, to generate the plasmid pIC20H/TK2.
[0116] Construction of pHOX1.4N/TK-TK2 proceeded in five sequential
steps as depicted in FIG. 6. First a clone containing hox1.4
sequences was isolated from a genomic .lambda. library. The
.lambda. library was constructed by inserting EcoRI partially
digested mouse DNA into the .lambda.-DASH.RTM. (Stratagene) cloning
phage. The hox1.4 containing phage were identified by virtue of
their homology to a synthetic oligonucleotide synthesized from the
published sequence of the hox1.4 locus. Tournier-Lasserve, et al.
(1989), Mol. Cell Biol., 9, 2273. Second, a 9 kb SalI-SpeI fragment
containing the hox1.4 homeodomain was inserted into
Bluescribe.RTM.. Third, a 1 kb BglII fragment within the hox1.4
locus was replaced with the Neo.sup.r gene isolated from pMCl Neo,
creating the plasmid pHOX1.4N. Fourth, the XhoI-SalI fragment by
HSV-tk from pIC19R/TK was inserted into the SalI site of pHOX1.4N,
generating the plasmid pHOX1.4N/TK. Fifth, the SalI-SpeI fragment
from pHOX1.4N/TK was inserted into a SalI-XbaI digest of the
plasmid pIC20HTK2, generating the final product, pHOX1.4N/TK/TK2.
This vector was digested with SalI to form a linear PNS vector
which was transfected into mouse ES cells as described in Example
1. Positive-negative selection and the method of forming transgenic
mice was also as described in Example 1. Southern blots of somatic
cells demonstrate that the disrupted hox1.4 gene was transferred to
transgenic offspring.
EXAMPLE 3
Inactivation of Other Hox Genes
[0117] The methods described in Examples 1 and 2 have also been
used to disrupt the hox1.3, hox1.6, hox2.3, and int-1 loci in ES
cells. The genomic sequences for each of these loci (isolated from
the same-Dash library containing the hox1.4 clone) were used to
construct PNS vectors to target disruption of these genes. All of
these PNS vectors contain the Neo gene from pMCi-Neo as the
positive selection marker and the HSV-tk and HSV-tk2 sequences as
negative selection markers.
8TABLE V Other Murine Developmental Genes Inactivated by PNS
Neo-Insertion Locus Genomic Fragment Sequence Ref. Site hox1.3 11
kb Xba-HindIII Tournier-Iasserve, EcoRI-site in et al. (1989),
homeo-domain MCE, 9, 2273 hox1.6 13 kb partial RI Baron, et al.
(1987), BglII-site in EMBO, 6, 2977 homeo-domain hox2.3 12 kb BamHI
Hart, et al. (1987), BglII-site in Genomics, 1, 182 homeo-domain
int-1 13 kb BglII van Ooyen et al. XhoI-site in (1984), Cell, 39,
233 exon 2
EXAMPLE 4
Vascular Graft Supplementing Factor VIII
[0118] In this example, a functional factor VIII gene is targeted
by a PNS vector to the .beta.-actin locus in human endothelial
cells. When so incorporated, the expression of factor VIII is
controlled by the .beta.-actin promoter, a promoter known to
function in nearly all somatic cells, including fibroblasts,
epithelial and endothelial cells. PNS vector construction is as
follows: In step IA (FIG. 7A), the 13.8 kb EcoRI fragment
containing the entire human .beta.-actin gene from the
.lambda.-phage, 14TB (Leavitte, et al. (1984), Mol. Cell Bio., 4,
1961) is inserted, using synthetic EcorI/XhoI adaptors, into the
XhoI site of the TK vector, pIC-19-R/TK to form plasmid pBact/TK.
See FIG. 7A.
[0119] In step 1B (FIG. 7B), the 7.2 kb SalI fragment from a factor
VIII cDNA clone including its native signal sequence (Kaufman, et
al. (1988), JBC, 263, 6352; Toole, et al. (1986), Proc. Natl. Acad.
Sci. USA, 83, 5939) is inserted next to the Neo.sup.r gene in a
pMCI derivative plasmid. This places the neo.sup.r gene (containing
its own promoter/enhancer) 3' to the polyadenylation site of factor
VIII. This plasmid is designated pFVIII/Neo.
[0120] In step 2 (FIG. 7C), the factor VIII/Neo fragment is excised
with XhoI as a single piece and inserted using synthetic XhoI/NcoI
adaptors at the NcoI site encompassing the met-initiation codon in
pBact/TK. This codon lies in the 2nd exon of the .beta.-actin gene,
well away from the promoter, such that transcription and splicing
of the mRNA is in the normal fashion. The vector so formed is
designated pBact/FVIII/Neo/TK.
[0121] This vector is digested with either ClaI or HindIII which
acts in the polylinker adjacent to the TK gene. The linker vector
is then introduced by electroporation into endothelial cells
isolated from a hemophiliac patient. The cells are then selected
for G418 and gancyclovir resistance. Those cells shown by DNA
analysis to contain the factor VIII gene targeted to the
.beta.-actin locus or cells shown to express FVIII are then seeded
into a vascular graft which is subsequently implanted into the
patient's vascular system.
EXAMPLE 5
Replacement of a Mutant PNP Gene in Human Bone Marrow Stem Cells
Using PNS
[0122] The genomic clone of a normal purine nucleoside
phosphonylase (PNP) gene, available as a 12.4 kb, Xba-partial
fragment (Williams, et al. (1984), Nucl. Acids Res, 12, 5779;
Williams, et al. (1987), J. Biol. Chem., 262, 2332) is inserted at
the XbaI site in the vector, pIC-19-R/TK. The neo.sup.r gene from
pMCI-Neo is inserted, using synthetic BamHI/XhoI linkers, into the
BamHI site in intron 1 of the PNP gene. The linearized version of
this vector (cut with ClaI) is illustrated in FIG. 8.
[0123] Bone marrow stem cells from PNP patients transfected with
this vector are selected for neo.sup.r, gan.sup.r, in culture, and
those cells exhibiting replacement of the mutant gene with the
vector gene are transplanted into the patient.
EXAMPLE 6
Inactivation by Insertional Mutagenesis of the Hox 1.1 Locus in
Mouse ES Cells, Using a Promoterless PNS Vector
[0124] A promoterless positive selection marker is obtained using
the Neo.sup.R gene, excised at its 5' end by enzyme, EcoRI, from
the plasmid, pMCI-Neo. Such a digestion removes the Neo structural
gene from its controlling elements.
[0125] A promoterless PNS vector is used to insert the Neo gen into
the Hox 1/1 gene in ES cells. The Hox 1.1 gene is expressed in
cultured embryo cells (Colbarg-Poley, et al. (1985), Nature, 314,
713) and the site of insertion, the second exon, lies 3' to the
promoter of the gene (Kessel, et al. (1987), PNAS, 84, 5306;
Zimmer, et al. (1989), Nature, 338, 150). Expression of Neo will
thus be dependent upon insertion at the Hox 1.1 locus.
[0126] Vector construction is as follows:
[0127] Step 1--The neo gene, missing the transcriptional control
sequences is removed from pMCI-Neo, and inserted into the second
exon of the 11 kb, FspI-KpnI fragment of Hox 1.1 (Kessel, et al.
(1987), supra; Zimmer, et al. (1989), supra).
[0128] Step 2--The Hox 1.1-Neo sequences is then inserted adjacent
to the HSV-tk gene is pIC19R/TK, creating the targeting vector,
pHox1.1-N/TK. The linearized version of this vector is shown in
FIG. 9 This vector is electroporated into ES cells, which are then
selected for Neo.sup.r, GanC.sup.r. The majority of cells surviving
this selection are predicted to contain targeted insertions of Neo
at the Hox1.1 locus.
EXAMPLE 7
Inducible Promoters
[0129] PNS vectors are used to insert novel control elements, for
example inducible promoters, into specific genetic loci. This
permits the induction of specified proteins under the spatial
and/or temporal control of the investigator. In this example, the
MT-1 promoter is inserted by PNS int the Int-2 gen in mouse ES
cells.
[0130] The inducible promoter from the mouse metallothionein-I
(MT-I) locus is targeted to the Int-2 locus. Mice generated from ES
cells containing this alteration have an Int-2 gene inducible by
the presence of heavy metals. The expression of this gene in
mammary cells is predicted to result in oncogenesis and provides an
opportunity to observe the induction of the disease.
[0131] Vector construction is as follows:
[0132] Step 1--The Ecor1-BglII fragment from the MT-I gene
(Palmiter, et al. (1982), Cell, 29, 701) is inserted by blunt-end
ligation into the BSSHII site, 5' to the Int-2 structural gene in
the plasmid, pAT 153 (see discussion of Example 1).
[0133] Step 2--The MCI-Neo gene is inserted into the AvrII site in
intron 2 of the Int-2-MT-I construct.
[0134] Step 3--The int-2-MT-ILNeo fragment is inserted into the
vector, pIC 19R/TK, resulting in the construct shown in FIG.
10.
[0135] Introduction of this gene into mouse ES cells by
electroporation, followed by Neo.sup.r, GanC.sup.r, selection
results in cells containing the MT-I promoter inserted 5' to the
Int-2 gene. These cells are then inserted into mouse blastocysts to
generate mice carrying this particular allele.
EXAMPLE 8
Inactivation of the ALS-II Gene in Tobacco Protoplasts by PNS
[0136] A number of herbicides function by targeting specific plant
metabolic enzymes. Mutant alleles of the genes encoding these
enzymes have been identified which confer resistance to specific
herbicides. Protoplasts containing these mutant alleles have been
isolated in culture and grown to mature plants which retain the
resistant phenotype (Botterman, et al. (1988), TIGS, 4, 219;
Gasser, et al. (1989), Science, 244, 1293). One problem with this
technology is that the enzymes involved are often active in
multimer form, and are coded by more than one genetic locus. Thus,
plants containing a normal (sensitive) allele at one locus and a
resistant allele at another locus produce enzymes with mixed
subunits which show unpredictable resistance characteristics.
[0137] In this example, the gene product of the ALS genes
(acetolactate synthase) is the target for both sulfonylurea and
imidazolinone herbicides (Lee, et al. (1987), EMBO, 7, 1241).
Protoplasts resistant to these herbicides have been isolated and
shown to contain mutations in one of the two ALS loci. A 10 kb SpeI
fragment of the ALS-II gene (Lee, et al. (1988), supra; Mazur, et
al. (1987), Plant Phys., 85, 1110) is subcloned into the negative
selection vector, pIC-19R/TK. A neo.sup.r gene, engineered for
expression in plant cells with regulating sequences from the
mannopine synthase gene for the TI plasmid is inserted into the
EcoRI site in the coding region of the ALS-II. This PNS vector is
transferred to the C3 tobacco cell line (Chalef, et al. (1984),
Science, 223, 1148), carrying a chlorsulfuron.sup.r allele in
Als-I.
[0138] They are then selected for Neo.sup.r, GanC.sup.r. Those
cells surviving selection are screened by DNA blots for candidates
containing insertions in the ALS-II gene.
[0139] Having described the preferred embodiments of the present
invention, it will appear to those ordinarily skilled in the art
that various modifications may be made to the disclosed
embodiments, and that such modifications are intended to be within
the scope of the present invention.
Sequence CWU 1
1
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