JP2002522096A - Development of domain-specific genes - Google Patents

Abstract

(57) SUMMARY Compositions and methods for developing specific protein domains using recombinases and recombinant intermediates are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION In nature, the evolution (evolution) of genes and their encoded proteins occurs through a balance between recombination or mutation and selection. Although evolution in nature takes millions of years, methods and compositions for in vitro deployment have been developed to develop proteins with improved novel functions as a matter of time.

[0002] Current methods of in vitro gene development include random mutagenesis, or random cleavage and mixed polymerase chain reaction (P
(CR) repetition cycle. These methods combine multiple rounds of in vitro mutagenesis into a screening system to generate and identify the desired mutation or recombinant (Stemmer 1994. Nature 370: 389-391; Arnold 1996. Chemical Engineeri).
ng Science 51: 5091-5102). However, studies have shown that the desired mutations are likely to occur in regions or domains directly associated with function (Chen and Arnold. 1993.
AS USA 90: 5618-5622). In addition, to generate random mutations via a desired gene by these mutagenesis methods, it is necessary to screen a large number of undesired harmful mutants. This labor intensive and time consuming method is further complicated by the need for multiple subclonings and is very laborious if a selection system is not used due to the complexity of the screen system.

[0003] The definition of homologous recombination (HR) is the exchange of homologous or similar DNA sequences that exist between two DNA molecules. A very important feature of HR is that the enzyme responsible for the recombination event can pair with all homologous sequences as substrates. The ability of HR to transmit genetic information between DNA molecules makes targeted homologous recombination a very powerful method in genetic engineering and engineering. HR can be used to add subtle mutations at known sites, replace wild-type genes or gene segments, or introduce complete foreign genes into cells. However, the efficiency of HR is very low in living cells and depends on various parameters. The parameters include DNA
There is a method of delivery, its mode of packaging, its size and conformation, length and position of DNA in the sequence of target homology, efficiency of hybridization and recombination at chromosomal sites. Such variability severely limits the use of conventional HR for gene deployment in cell-based systems (Kucherlapati
et al., 1984. PNAS USA 81: 3153-3157; Smithies et al. 1985. Nature 317: 2
30-234; Song et al. 1987. PNAS USA 84: 6820-6824; Doetschaman et al. 1987.
Nature 330: 576-578; Kim and Smithies. 1988.Nuc. Acids. Res. 16: 8887-89
03; Koller and Smithies. 1989. PNAS USA 86: 8932-8935; Shesely et al. 199
1. PNAS USA 88: 4294-4298; Kim et al. 1991. Gene 103: 227-233).

[0004] The presence of recombinase activators in cell lines and cell-free systems has been
Significantly increase the frequency of use. Various proteins or purified extracts that promote HR (ie, recombinase activity) have been identified in prokaryotes and eukaryotes (Cox and Lehman., 1987. Annu. Rev. Biochem. 56: 229-262; Radding. 1
982. Annual Review of Genetics 16: 405-547; McCarthy et al. 1988. PNAS
USA 85: 5854-5858). These recombinases facilitate one or more steps in homologous pairing intermediate formation, strand exchange and / or other steps. The most studied recombinase at present is the E. coli RecA recombinase, which is involved in homology searches and strand exchange reactions (Cox and Lehman, 1987, supra).

[0005] The bacterial RecA protein (Mr37,842) catalyzes homologous pairing and strand exchange between two homologous DNA molecules (Kowalczykowski et al. 1994.
Microbiol. Rev. 58: 401-465; West. 1992. Annu. Rev. Biochem. 61: 603-640.
Roca and Cox. 1990. CRC Cit. Rev. Biochem. Mol. Biol. 25: 415-455; Rad.
ding. 1989. Biochim. Biophys. Acta. 1008: 131-145; Smith. 1989. Cell 58: 8
07-809). The RecA protein is a RecA protein monomer with a stoichiometry of 1 for every 3 to 4 nucleotides contained in the DNA and co-bonds with all predetermined sequences of single-stranded DNA (Cox and Lehman, 1987, supra). . With this combination,
A unique right-handed helical nucleoprotein filament is formed in which the length of DNA is extended by a factor of 1.5 (Yu and Egelman. 1992. J. Mol. Biol. 227: 33).
4-346). These nucleoprotein filaments, referred to as DNA probes, are important "central drivers of homology search" and catalyze DNA pairing. When the filament finds a target gene sequence that is homologous to itself, the DNA probe strand invades the target and forms a hybrid DNA structure. This is called the junction molecule or D loop (D
NA replacement loop) (McEntee et al. 1979. PNAS USA 76: 2615-2619; Sibata
et al. 1979. PNAS USA 76: 1638-1642). The phosphate groups of the DNA inside the RecA nucleoprotein filament are protected from digestion by phosphodiesterase and nuclease.

[0006] The RecA protein is the prototype of a universal class of recombinase enzymes that facilitate probe-target pairing reactions. Recently, a gene homologous to E. coli RecA (Ra
d51 protein family) have been isolated from all eukaryotic communities, including yeast and humans. RAD51 protein is obtained in the same manner as RecA protein.
Promotes homologous pairing and invasion and exchange of strands between homologous DNA molecules (Sung. 1
994.Science 265: 1241-1243; Sung and Robberson. 1995.Cell 82: 453-461; G
upta et al. 1997. PNAS USA 94: 463-468; Baumann et al. 1996. Cell 87: 757-
766).

[0007] Methods and compositions used to target and alter by homologous recombination, substitution, insertion, deletion are described below. U.S. Patent Application No. 08/38
1634; 08/882756; 09/301153; 08/781329; 09/288586; 09/209676; 09/007020;
09/179916; 09/182102; 09/182097; 09/181027; 09/260624; and International Patent Application Nos. US97 / 19324; US98 / 26498; US98 / 01825.

[0008] It is therefore an object of the present invention to provide an efficient method for the development of domain-specific genes that yields the greatest diversity and increases the probability of identifying the desired gene.

(Summary of the Invention) The present invention provides a method for developing a domain-specific gene. The method comprises forming a plurality of recombinant intermediates comprising a target nucleic acid sequence encoding a desired amino acid sequence, a recombinase, a plurality of targeting polynucleotides. The polynucleotides for targeting are substantially complementary to each other and each include a homology clamp that substantially corresponds to or is substantially complementary to the intended sequence of the target nucleic acid, and may be random or random. Contains degenerate sequences. The expected sequence encodes a domain of the amino acid sequence. The method involves contacting the intermediate with a recombinant competent cell to create a library of modified target nucleic acids. The modified target nucleic acid sequence is expressed in a cell to create a pool of variant amino acid sequences. The method further includes selecting and isolating cells containing the modified target nucleic acid that has expressed the variant amino acid having the desired activity.

In another aspect of the invention, a domain-specific gene expansion method comprises forming a recombinant intermediate comprising a target nucleic acid sequence encoding a desired amino acid sequence, a recombinase, and a paired targeting polynucleotide. . The targeting polynucleotides include homology clamps that are substantially complementary to each other and that each substantially correspond to or are substantially complementary to the intended sequence of the target nucleic acid. The expected sequence encodes a domain of the amino acid sequence. The method further comprises contacting the intermediate with a single-strand-specific or ligation-specific nuclease to form a truncated or open-ended target nucleic acid. The region hybridized or adjacent to the junction is nuclease sensitive. The target nucleic acids are reassociated and recombined to create a library of modified target nucleic acids. The target nucleic acid sequence is expressed to create a pool of variant amino acid sequences. The variant amino acids are selected and characterized to identify an altered target nucleic acid encoding the desired variant amino acid sequence.

In yet another aspect, each method is repeated one or more times to further develop variant amino acid sequences having the desired activity. In another embodiment, one or more domains or proteins are deployed simultaneously.

DETAILED DESCRIPTION OF THE DRAWINGS FIG. 1 shows domain specific gene deployment (DSGE) by recombinase-mediated complementary single-stranded DNA targeting (cssDNA or targeting polynucleotide). 1) Labeling of domain B of the desired gene with RecA-coated cssDNA denaturing probe, 2) Deproteinization and transformation of multi-stranded recombinant DNA hybrid product in bacteria, 3) Screening and selection of desired mutant 4) Repeat steps 1-3.

FIG. 2 shows the natural gene targeting assay (dual D-loop formation). cssDN
A is made by heat denaturation of the double-stranded DNA PCR product and
Coated with cA nucleoprotein (represented by o) to form nucleoprotein filaments. RecA nucleoprotein filaments can be added sequentially or simultaneously to the duplex to target homologous sites in this natural DNA. Interaction of one filament with the double helix forms a triple stranded DNA intermediate (single D loop). It is unstable after deproteinization. The addition of the complementary strand renders the otherwise unstable intermediate more stable through the formation of a kinetic trapped double D-loop. This double D loop is highly stable after deproteinization, can be handled in a number of different ways, and is biologically active.

FIGS. 3A-B show probe target hybrid uplifting homologous recombination in bacteria. Panel A: Schematic for the EHR assay used in Panel B. Panel B
: CssDNA probe in E. coli: Enhanced homologous recombination of target hybrid (EH
The data for R) is shown. Homologously targeted probes: Target hybrids increase the frequency of homologous recombination in competent cells. CssDNA probe formed: Target hybrid was introduced into RecA + and RecA-E. Coli. The molar ratio of cssDNA probe: target hybrid in the in vitro targeting reaction is
It was 1: 1 to 1: 5.6. % Recombinant / total colonies is the percentage of blue colonies in the total population of ampicillin resistant colonies. The 0% recombination group did not produce any blue colonies in at least 105 plate colonies. Plasmid DNA was isolated from blue colonies grown continuously for three generations, and it was examined whether homologous recombination occurred stably in the lacZ gene.

FIG. 4 shows the design of a DSGE DNA probe. DSGE DNA probes can target either a single domain or multiple domains and cause mutations. These recombinant DSGE DNAs may contain mutations in their full length or may be denatured. The resulting recombinant or variant contains many possible combinations for mutations in the target domain. In this case, there is targeting and mutagenesis of the two domains, and the RecA protein is unclear. Single domain shuffling probes target and mutate a single region. These probes contain two external homology clamps whose sequence exactly corresponds to the sequence in the target. The internal region has varying degrees of mismatch and mutates and shuffles this specific region.

FIG. 5 shows a targeting polynucleotide for deploying the CDR region of the scFv to botulinum neurotoxin.

FIG. 6 shows a flow chart of steps in the development of a single-chain scFv antibody phage library by DSGE. Both light and heavy chain variable regions are present in one molecule in the single-chain Fv. Transformation of CDR region target hybrids into helper phage in E. coli allows for inclusion and expression. After performing a screen to remove undeployed antibody-phage, the method is repeated to extend the antibody-phage library.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides methods and compositions for domain-specific gene deployment.
In one embodiment of the invention, the method comprises targeting a nucleic acid sequence encoding a specific protein domain to effect modification of a plurality of target sequences. That is, by targeting the recombination probe of the present invention to a specific protein domain, a specific expansion known to be or is believed to retain gene deployment and selection in a specific activity or function. Target against the domain. This method creates the greatest diversity in the desired specific domains, thus reducing the size of the mutation library to be screened and improving or increasing the likelihood of finding genes with the desired attributes . Thus, the libraries of the present invention are rich in advantageous mutant or recombinant sequences.

The present invention involves combining a plurality of pairs of single-stranded targeting polynucleotides, a predetermined target nucleic acid, and a recombinase to form a polynucleotide: target nucleic acid complex. The targeting polynucleotide comprises at least one homology clamp and a random or degenerate sequence to target a predetermined domain of the target nucleic acid.
Complex with multiple recombination-competent cells (catalyzes strand exchange or homologous recombination intracellularly)
Into a library of modified nucleic acids. Cells containing the modified nucleic acid encoding the polypeptide of the desired properties are selected and isolated. This method is preferably repeated to further develop the desired target domain. This is shown schematically in FIG.

In another aspect of the present invention, there is provided a method of domain-specific DNA truncation for domain-specific gene deployment. The method includes combining a pair of single-stranded targeting polynucleotides, a target nucleic acid of interest, and a recombinase to form a polynucleotide: target nucleic acid complex. The targeting polynucleotides are substantially complementary to each other and include at least one homology clamp for targeting a predetermined domain of the target nucleic acid. The polynucleotide: target nucleic acid complex is treated with a single strand specific nuclease. This nuclease preferentially cleaves the region flanking the polynucleotide: target nucleic acid complex region (Ferrin and Camerini-Otero, 1991, Scien
ce, 254 1494-1497). That is, in the complex, the domain is protected from recombination by the initial presence of the recombinase. The nuclease is inactivated and the complex is dissociated. The cleaved target nucleic acids are reassociated and recombined with the PRC to create a library of nucleic acids with preferential modifications in the cleavage region. A library of modified nucleic acids can be introduced into host cells and expressed. Cells containing the modified nucleic acid encoding the polypeptide of the desired properties are selected and isolated. This method is preferably repeated to further develop the desired target domain.

In each of the above methods, a single domain or multiple domains by selection are targeted. The above methods and compositions can be selectively used in combination for domain-specific gene deployment. For example, one or more domain-specific DNA breaks
One or more domain-specific expansions are carried out using the above-mentioned plurality of target polynucleotides.

[0022] The method of the present invention does not require multiple subcloning steps. This is of particular relevance for large complex vectors, such as lambda, BACS, PACS, YACS,
This is the case when using genomic DNA such as MACS, and when the multiple subcloning step results in mutagenesis and shuffling of unique sites in large vectors is particularly long and time consuming.

Accordingly, the present invention provides a method of introducing a recombination-forming probe or hybrid complex into recombination-competent cells and linking them to in vitro and in vivo recombination and expansion methods. By creating homologous recombination intermediates in vitro, a panel or library of mutated and shuffled genes is created for in vitro development. When linked to an in vivo system, an evolved gene encoding a protein of the desired properties can be selected in vivo.

The present invention can be used in various important ways. First, these methods can be used to create transformants, animal and plant models for disease. Thus, for example, the domain-specific targeting polynucleotide used in the homologous recombination method can produce an animal having a wide range of mutations in a wide range of functionally relevant genes. It can result in a wide range of phenotypes, including those associated with disease states. This can be done at the cellular level, identifying genes involved in the cellular phenotype. That is, target identification. Second, domain targeting can be used on diseased or modified cells or animals. Briefly, domain targeting can be performed to identify “inverted genes”. This gene can regulate disease states caused by different genes, either the same gene family or a completely different gene family. Also, for example, deletion of one type of enzymatic activity results in a disease phenotype, which can be compensated for by altering different but homologous enzymatic activities.

In addition, the method can be used to create a library of modified nucleic acids containing extra chromosomal sequences. It can be expressed in cells to create a library of modified proteins and screened for some useful and interesting properties. This property includes, but is not limited to, increased and altered stability (to heat, pH, oxidation, proteases, etc.), altered binding, altered activity, desirable alterations in properties such as immunogenicity.

Accordingly, the present invention provides a method for homologous recombination. As used herein, “homologous recombination” (HR) refers to the exchange of homologous or similar DNA sequences between two DNA molecules. A fundamental property of HR is that the enzyme responsible for the recombination event can pair homologous sequences as substrates. HR that can transfer genetic information between DNA molecules
Makes the targeted homologous recombination a very powerful method in genetic engineering and engineering. HR can be used to insert, remove and / or replace one or more nucleotides in a gene or a segment thereof, or to introduce or remove a gene in a target nucleic acid.

[0027] Once the protein domains have been identified, the compositions of the invention can be made. The composition of the present invention is a composition comprising at least one recombinase and at least two single-stranded targeting polynucleotides. These polynucleotides are substantially complementary to each other, and each has a domain homology clamp for the gene family.

“Recombinase”, when included with an exogenous targeting polynucleotide, measures the frequency of recombination and / or localization between the targeting polynucleotide and the intended endogenous DNA sequence. A protein or peptide (eg, L2 peptide) that is increased as much as possible. That is, in a preferred embodiment, the increase in recombination frequency is usually in the range of 10 ^-8 to 10 ^-4 to 10 ^-4 to 10 ^1, preferably 10 ^-3 to 10 ^1, and most preferably 10 ^-2 to 10 ⁰ Is achieved.

In the present invention, recombinases are a family of RecA-like and Rad51-like recombinant proteins with essentially all or most of the same functions. In particular, (i) the ability of the recombinase protein to properly bind and position the targeting polynucleotide on the homologous target, and (ii) efficiently find and bind to the complementary endogenous sequence Recombinase protein / target polynucleotide complex. The best characterized RecA protein is derived from E. coli and a number of mutant RecA proteins have been elucidated in addition to the wild-type protein (eg, recA803, see Madiraju et al., PNAS U).
SA 85 (18): 6592 (1988); Madiraju et al., Biochem. 31: 10529 (1992); Lav
ery et al., J. Biol. Chem. 267: 20648 (1992)). In addition, many organisms have RecA-like recombinases capable of strand transfer (eg, Fugisawa et al., (19)
85) Nucl. Acids Res. 13: 7473; Hsieh et al., (1986) Cell 44: 885; Hsieh
et al., (1989) J. Biol. Chem. 264: 5089; Fishel et al., (1988) Proc. Nat.
l. Acad. Sci. (USA) 85: 3683; Cassuto et al., (1987) Mol. Gen. Genet. 20
8:10; Ganea et al., (1987) Mol.Cell Biol. 7: 3124; Moore et al., (1990
) J. Biol. Chem. 19: 11108; Keene et al., (1984) Nucl. Acids Res. 12: 30.
57; Kimeic, (1984) Cold Spring Harbor Symp. 48: 675; Kmeic, (1986) Cell
44: 545; Kolodner et al., (1987) Proc. Natl. Acad. Sci. USA 84: 5560; Sug
Natl. Acad. Sci. USA 85: 3683; Halbrook et al., (1989) J. Biol. Chem. 264; 21403; Eisen et al., (1988) Proc. Natl. . Acad
Sci. USA 85: 7481; McCarthy et al., (1988) Proc. Natl. Acad. Sci. USA
85: 5854; Lowenhaupt et al., (1989) J. Biol. Chem. 264; 20568, incorporated herein by reference.

[0030] Examples of such recombinase proteins include, but are not limited to, RecA,
RecA803, uvsX, other RecA mutations and RecA-like recombinase (Roca, AI (1990) Crit. Res. Biochem. Molec. Biol. 25: 415), sep.
1 (Kolodner et al. (1987) Proc. Natl. Acad. Sci. (USA) 84: 5560; Tishko
ff et al. Molec Cell Biol. 11: 2593), RuvC (Dunderdale et al. (1991) Na
354: 506), DST2, KEM1, XRN1 (Dykstra et al. (1991) Mol
ec. Cell. Biol. 11: 2583), STPα / DST1 (Clark et al. (1991) Mole.
c. Cell. Biol. 11: 2576), HPP-1 (Moore et al. (1991) Proc, Natl. A.
cad. Sci. (USA) 88: 9067), other target recombinases (Bishop et al. (1992)
Cell 69: 439; Shinohara et al. (1992) Cell 69: 457, which is hereby incorporated by reference).

RecA was obtained from E. coli strains, JC12772 and JC15369 (AJ Clark an
d M. Madiraju, obtained from the University of California, Berkeley or commercially available). These strains contain the RecA coding sequence on a "running" replicating plasmid vector that is present at high copy numbers per cell. RecA803 protein is a highly active mutant of wild-type RecA. Several examples of recombinase proteins are known, for example, proteins derived from cells of Drosophila, yeast, human, non-human mammals, and having biological properties similar to RecA (ie,
RecA-like protein), for example, Rad51, Rad57, dmel from mammals and yeast. In addition, recombinases may actually be a complex of proteins, or "recombinosomes." Included within the definition of recombinase are portions or fragments of the recombinase that retain the biological activity of the recombinase, and variants or variants of the wild-type recombinase that retain the biological activity, for example, an E. coli RecA803 variant that has high recombinase activity. .

In a preferred embodiment, RecA and rad51 are used. For example, Rec
The A protein can be commonly obtained from strains that overproduce this protein. Wild-type E. coli RecA protein and mutant RecA803 protein can be purified from such strains. Alternatively, RecA can be purchased.
For example, Pharmacia (Piscataway, NJ) or Boehringer Mannheim (India
napolis, Indiana).

RecA and its homologs form a nucleoprotein fragment, which is a single-stranded DN
A is coated. In this nucleoprotein fragment, one monomer of the RecA protein binds to about three nucleotides. While this property of RecA coating single-stranded DNA is essentially sequence-independent, certain sequences facilitate the initial retention of RecA in a polynucleotide (eg, a nuclear-retaining sequence). Nucleoprotein fragments can be formed on essentially any DNA and can be formed in cells,
A single-stranded and double-stranded complex is formed, but the retention conditions for dsDNA are s
Somewhat different from sDNA.

[0034] The recombinase binds to the targeting polynucleotide. The outline is further described below. “Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammatical equivalents means at least two nucleotides covalently linked to each other. The nucleic acids of the present invention generally contain a phosphodiester bond, but in some cases also include nucleic acid analogs, which are separated by another backbone, such as phosphoramide (Beaucage e).
t al., Tetrahedron 49 (10): 1925 (1993) and quotations; Letsinger. J. Org.
Chem. 35: 3800 (1970); Sprinzl et al., Eur. J. Biochem. 81: 579 (1977);
Letsinger et al., Nucl. Acids Res. 14: 3487 (1986); Sawai et al., Chem.
Lett. 805 (1984), Letsinger et al., J, Am, Chem. Soc. 110: 4470 (1988):
Pauwels et al., Chemica Scripta 26: 141 (1986)), phosphorothioates,
Phosphorodithioate, O-methyl phosphoramidite linkage (see, Eckstein,
Oligonucleotides and Analogues: A Practical Approach, Oxford University
Press), peptide nucleic acid scaffolds and linkages (see Egholm, J. Am. Chem. Soc. 1
14: 1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31: 1008 (1992); Nie
lsen, Nature, 365: 566 (1993); Carlsson et al., Nature 380: 207 (1996),
All of which are hereby incorporated by reference). These modifications to the ribose-phosphate backbone or base result in the chemical components (2′O-methyl and 5 ′
Facilitate the addition of other moieties, such as' modified substituents, described below) or increase the stability and half-life of such molecules in a physiological environment.

[0035] The nucleic acid can be a single- or double-stranded sequence, or can contain portions of a single- or double-stranded sequence. Nucleic acids can be DNA, genomic and cDNA, RNA or hybrids. In a hybrid, the nucleic acid contains a combination of deoxyribo and ribonucleotides, and any combination of bases. Bases include uracil, adenine, thymine, cytosine, guanine, inosine, xanthanine, hypoxatanin and the like. Thus, for example, chimeric DNA-RNA molecules can be used (Cole-Strauss et al., Science 273: 1386 (1996) and Y
oon et al., PNAS USA 93: 2071 (1996), both of which are hereby incorporated by reference).

In general, a targeting polynucleotide can include any number of structures, as long as the functionality of the targeting polynucleotide that results in homologous recombination does not change. For example,
Alternative structures of recombinase coating can still occur.

[0037] As used herein, "targeting polynucleotide" refers to a polynucleotide used to make alterations in the protein domains described herein. The targeting polynucleotide is generally ssDNA or dsDNA, most preferably two complementary single-stranded DNAs.

The targeting polynucleotide is generally at least about 5 to 2000 nucleotides in length, preferably about 12 to 200 nucleotides in length, at least about 200 to 50 nucleotides in length.
0 nucleotides, more preferably at least about 500 to 2000 nucleotides or longer; however, the length of the targeting polynucleotide is about 20,0
Increasing from 00 to more than 50,000 to 400,000 nucleotides decreases the efficiency of delivery of the intact targeting polynucleotide to the cell. The length of homology can be selected at the discretion of the practitioner, based on the sequence composition and complexity of the intended endogenous target DNA sequence, and the guidance provided in the art, and is generally higher when using non-recombinase-mediated methods. The homology of the segments from 1.3 to 6.8 kilobases is indicated (Hasty et al. (1991) Molec. Cell. Biol. 11: incorporated herein by reference).
5586; Shulman et al. (1990) Molec. Cell. Biol. 10: 4466).

The targeting polynucleotide has at least one sequence substantially corresponding to or substantially complementary to the intended endogenous DNA sequence. As used herein, “predetermined target nucleic acid”, “predetermined target sequence”, “predetermined target nucleic acid domain” means a polynucleotide sequence contained in a target nucleic acid. Such sequences include, for example, chromosomal sequences (eg, structural genes, regulatory sequences containing promoters and enhancers, recombination hotspots, repeat sequences, integrated proviral sequences, hairpins, palindromes), chloroplasts and mitochondrial D
Episomal or extrachromosomal sequences containing NA sequences (eg, replicable plasmids,
Virus replication intermediate). "Scheduled" or "preselected" means that the target sequence can be selected at the practitioner's intent based on known or predicted sequence information and can be selected by a specific site-specific recombinase (eg, FLP recombinase or CRE recombinase). It means that you are not restricted to the recognized specific site. The intended endogenous DNA target sequence in some embodiments is other than a naturally occurring germline DNA sequence (eg, a transgene, parasitic, mycoplasmal, viral sequence). An endogenous polynucleotide is a polynucleotide that is transferred into a target cell but is not replicated in a host cell. For example, a viral genomic polynucleotide that enters a cell by fusion of a virion with the cell is an exogenous polynucleotide, whereas a copy of a subsequently produced viral polynucleotide copy in an infected cell is an endogenous sequence (and For example, integrated into the cell chromosome).
Similarly, transgenes microinjected or transferred into cells are exogenous polynucleotides, whereas transgene integration and replication of replication are endogenous sequences.

[0040] In a preferred embodiment, the target nucleic acid comprises a nucleotide sequence that encodes a protein or polypeptide. However, as outlined herein, target nucleic acids can also be made in non-coding regions. As used herein, “protein” refers to at least 2
Means two covalently linked amino acids and includes proteins, polypeptides, oligopeptides and peptides. As used herein, "amino acid" or "peptide residue" refers to naturally occurring and naturally modified amino acids. For example, "amino acids" include imino acid residues such as proline and hydroproline. "Naturally modified amino acids" include, for example, amino acids modified to contain carbohydrate structures, such as high mannose and complex carbohydrates, phosphate groups, and lipids. In a preferred embodiment, the amino acids are in the (S) or L configuration.

The nucleotide sequence encoding the polypeptide is preferably operatively linked to a transcription / translation control element capable of functioning in the desired host cell, and expresses the encoded protein upon introduction of the target nucleic acid. Transcription control elements include constitutive or inducible elements. When the desired host cell is a eukaryotic cell, a henhancer element is optionally used. In a preferred embodiment, the target nucleic acid is a viral vector such as a retrovirus, phage, genomic and chromosomal DNA.
This is the type of NA such as BAC, PAC, YAC, and MAC.

As used herein, “naturally occurring” with respect to a subject means that the subject can be naturally occurring. For example, polynucleotide sequences that can be isolated from natural sources and present in organisms (including viruses) that have not been intentionally modified by humans are naturally occurring.

The method of the present invention is used for modification and expansion of a protein domain. That is, in a preferred embodiment, the target nucleic acid comprises a nucleic acid encoding a protein domain. As used herein, “protein domain” and grammatical equivalents thereof refer to a region of a protein that provides specific structural and / or functional properties. Thus, a protein domain is an enzyme active site, ligand binding site, extra-active center effector region, epitope, region of a protein (modified by the addition of carbohydrates, phosphate groups, lipids). Domains also include extracellular, intercellular, and transmembrane domains, with respect to hydrophilicity or hydrophobicity of the region. Cell targeting sequences, such as signal peptides, nucleic acid localizing sequences, mitochondrial localizing sequences, direct proteins to localization at the extracellular or subcellular level, and are domains. Additional domains include regions of the protein that interact with other proteins or nucleic acids, such as multiplexed sequences, zinc finger motifs, and the like. In other embodiments,
A protein domain is a region encoded by an exon.

The polynucleotide for targeting has at least one sequence substantially corresponding to or substantially complementary to the target nucleic acid. That is, in a preferred embodiment,
Corresponds to or is complementary to a nucleic acid encoding a protein domain. As used herein, "corresponding" means that the polynucleotide sequence is homologous to all or a portion of a reference polynucleotide sequence (i.e., similar or identical and may not be strictly evolutionarily related). ) Or that the polypeptide sequence is identical to the reference polypeptide sequence. On the other hand, the term "complementary" is used herein to mean that the complementary sequence is capable of hybridizing to all or part of a control polynucleotide sequence. Thus, one of the complementary single-stranded targeting polynucleotides is complementary to one strand of the endogenous target domain sequence (ie, Watson).
, Corresponding to the other strand of the endogenous target domain sequence (ie, click). in this way,
The complementarity between two single-stranded targeting polynucleotides need not be perfect. For example, the nucleotide sequence "TATAC" corresponds to the control sequence "TATAC" and is completely complementary to the control sequence "GTATA".

As used herein, “substantially corresponding” or “substantial identity” or “
The term "homology" refers to a nucleic acid sequence that has at least 50% sequence identity as compared to a control sequence, typically at least about 70% sequence identity as compared to a control sequence, and preferably at least about 85%. A feature of nucleic acid sequences having sequence identity.
Percent sequence identity is calculated excluding small deletions or additions of less than 25% in total of control sequences. A control sequence can be a subset of a large sequence, such as a portion of a gene or flanking sequence or a repeated portion of a chromosome. However, a control sequence is at least 18 nucleotides in length, typically at least about 30 nucleotides in length, and preferably at least about 50 to 100 nucleotides in length. As used herein, "substantially complementary" is used to refer to a sequence that is substantially complementary to a sequence that substantially corresponds to a control sequence. In general, targeting efficiency increases with the length of the targeting polynucleotide portion that is substantially complementary to the control sequence present in the target DNA.

As used herein, “sequence homology” refers to sequence similarity or sequence identity. Nucleic acid similarities are described, for example, in BLASTIN (Altschul et al. 1990. j. Mol. Bio.
l. 147: 195-197). BLASTIN is a simple scoring system with +5 pairings and -4 mispairings. To gain computer efficiency,
Invalid parameters are directly embedded in the source code.

In another embodiment, the percent nucleic acid sequence homology is determined. In the calculation of% homology,
No relative weighting is performed on the various types of sequence variation, eg, insertions, deletions, substitutions, and the like. Only the identity is defined as positive (+1), and all types of sequence variation are defined as 0. This eliminates the need for emphasis or parameters as described above for sequence similarity calculations. To calculate% sequence identification, for example, the number of matching identical residues in the equilibrium region is divided by the number of "shorter" sequence residues and multiplied by 100. "Longer" sequences are those that have the most active residues in the equilibrium region.

These corresponding / complementary sequences are sometimes referred to herein as “domain homology clamps” because they serve as templates for homologous pairing with the intended endogenous sequence. Thus, a "domain homology clamp" is a portion of a targeting polynucleotide that can specifically hybridize to a nucleic acid encoding a domain within a gene of interest. “
"Specific hybridization" is contemplated herein as a targeting polynucleotide (e.g., a polynucleotide of the invention that may contain substitutions, deletions and / or additions as compared to a predetermined target nucleic acid sequence). The target polynucleotide hybridizes preferentially to the intended target nucleic acid, for example, in a Southern blot of nucleic acid prepared from target cells containing the target nucleic acid sequence, At least one separate band can be identified and / or the targeting polynucleotide in the intact nucleus becomes localized for distinct chromosomal locations characterized by unique or repetitive sequences. like,
Target Domain Functional domain sequences are present in one or more target polynucleotide species (eg, a particular target sequence can occur in multiple members of a gene family). Obviously, optimal hybridization conditions will vary depending on the sequence composition and length of the targeting polynucleotide and target, and the experimental method chosen by the practitioner. Various guidelines, which are hereby incorporated by reference, may be used in the selection of appropriate hybridization conditions (Maniatis et al., Mo.
lecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor
, NY and Berger and Kimmel, Methods in Enzymology, Volume 152, Guide
to Molecular Cloning Techniques (1987), Academic Press, Inc.San Diego,
CA). Methods for hybridizing targeting polynucleotides to discrete chromosomal locations in the intact nucleus are known in the art, for example, WO 93/05177 and Kowa
See lczykowski and Zarling (1994), Gene Targeting, Ed. Manuel Vega.

In the targeting polynucleotide, such domain homology clamps are typically located at or near the 5 ′ or 3 ′ end, preferably the domain homology clamps are endogenous or each of the polynucleotides. Terminally located (Berinstein et al. (1992) Molec. Cell. Biol. 12: 360, incorporated herein by reference).
. Without being bound by a particular theory, the addition of recombinase is short (ie, about 1
A targeting polynucleotide having a homologous segment,
And target polynucleotides with long homologous segments would allow for efficient gene targeting.

Thus, it is preferred that the targeting polynucleotides of the invention have a domain homology clamp that is highly homologous to the intended target endogenous domain functional domain nucleic acid sequence. Typically, a targeting polynucleotide of the invention will comprise from about 18 to 35
Preferably, the domain homology clamp has at least one domain homology clamp that is at least about 20 to 100 nucleotides in length, more preferably at least about 100-500 nucleotides in length. The degree of sequence homology between the base composition of the sequence and the target sequence is
Determine optimal and minimum clamp lengths (eg, GC-rich sequences are typically thermodynamically stable and generally require short clamp lengths). For this,
Although both domain homology clamp length and sequence homology are determined only in reference to the specific intended sequence, domain homology clamps are generally at least about 10 nucleotides in length and correspond substantially to the intended target sequence. Or must be substantially complementary. Preferably, the homology clamp is at least about 10, preferably at least about 50 nucleotides in length, and is substantially identical or complementary to the intended target sequence. Without being bound by a particular theory, the addition of the recombinase to the targeting polynucleotide can be between homologous and non-isogenic sequences (eg, Bal
between the exon 2 sequence of the albumin gene of b / c mice and the exon 2 sequence of the homologous albumin gene of C57 / BL6 mice), and the homologous recombination between isogenic sequences.

In one embodiment of the invention, the targeting polynucleotide comprises a plurality of targeting polynucleotides comprising at least one partitioning homology clamp and a degenerate sequence. As used herein, “plurality” means two or more. The targeting polynucleotide can be used for mutagenesis and expansion of the target nucleic acid sequence. This sequence encodes a specific protein domain by insertion, deletion and / or substitution of a nucleic acid sequence encoding the domain. In one embodiment, if the degenerate sequence is completely random, all combinations of nucleotides are possible. In another embodiment, the degenerate sequence is biased, for example, to eliminate the coding sequence for the copy / translation stop signal. In another embodiment, the degenerate sequence is biased to display the codon bias of the host cell or class of the organism. Selectively biasing the degenerate sequence,
Randomize special arrays while keeping other arrays constant. The length of the degenerate sequence is
Determined by the practitioner and based on the desired number of nucleotides in the sequence to be modified.

[0052] In another embodiment, the targeting polynucleotide is substantially identical to the intended target sequence. In the presence of the recombinase, the targeting polynucleotide forms a complex with the intended target sequence of the target nucleic acid. As part of the complex, the intended target sequence is resistant to nuclease digestion. Polynuclease: The region flanking the target complex is sensitive to single-strand-specific exonuclease. Thus, to affect domain-specific expansion, these regions are cut and the resulting fragments are reassociated with RCP and recombined. This method is described below and in addition to Stemmer et al. Nature.
0: 389-391 and Stemmer et al. PNAS USA 91: 10747-10751, which are hereby incorporated by reference.

The formation of a heteroduplex bond is not a stringent method. That is, according to genetic evidence, the classical phenomenon of meiotic gene conversion and aberrant meiotic segregation
Partly due to the inclusion of mismatched base pairs at the heteroduplex junction and some correction of these mismatched base pairs before replication. Observation with RecA protein
Affects the identification of perfect or near perfect homology correlations and provides information on the parameters involved in mismatched base pairs at heteroduplex junctions. The ability of the RecA protein to drive strand exchange through all single-stranded pair mismatches, and to form wide mismatch contacts in superhelical DNA, suggests a role for this protein in recombination and gene conversion. Express. This error-prone process may also be related to the role of this protein in mutagenesis. Homologous recombinants have been obtained in RecA-mediated pairing reactions involving DNA of Φ × 174 and G4 that are approximately 70% homologous (Cunningham et al. (1981) Cell 24: 213), but the predominant RecA form is Forms a homologous bond with the highly homologous sequence and is involved in mediating the homology search between the invading DNA strand and the recipient DNA strand, and is a relatively stable heteroduplex in highly homologous regions. To produce

Thus, in fact, recombinases can drive homologous recombination reactions between striking but not complete homologous strands. This reaction allows for gene conversion and modification of the target sequence. Thus, the targeting polynucleotide expresses the nucleotide substitutions, insertions and deletions in the endogenous common functional domain nucleic acid sequence, and thus the corresponding amino acid substitutions, insertions and deletions from the endogenous common functional domain nucleic acid sequence. Can be inserted into a protein. As used herein, “endogenous” refers to a naturally occurring sequence, ie, a sequence or substance derived from within a cell or organism. Similarly, "exogenous" refers to a sequence or substance derived from outside a cell or organism.

In a preferred embodiment, two substantially complementary targeting nucleotides are used. In one embodiment, the targeting polynucleotide that can be coated with a recombinase forms a double-stranded hybrid, but when the recombinase is RecA,
Loading conditions can be somewhat different from those using single-stranded nucleic acids.

In a preferred embodiment, two substantially complementary single stranded targeting polynucleotides are used. The two complementary single-stranded targeting polynucleotides are usually of equal length, but this is not required. However, as described below, the stability of the four-stranded hybrids of the invention is presumed to be related, in part, to the loss of significant non-hybridized single-stranded nucleic acids, and therefore significant unpaired sequences are not preferred. . Further, as described above,
The complementarity between two targeting polynucleotides need not be perfect. The two complementary single-stranded targeting polynucleotides will bind the intended endogenous target sequence, generally
Simultaneously or simultaneously insert into a target cell carrying with at least one recombinase protein (eg RecA). Under most circumstances, the targeting polynucleotide is incubated with RecA or another recombinase prior to insertion into the target cell, and the recombinase protein is "loaded" into the targeting polynucleotide and coated with the nucleic acid as described below. Preferably. Incubation conditions for such recombinase loading are described below and also in US application Ser. No. 07 / 755,462, filed Sep. 4, 1991; No. 07 / 910,791, filed July 9, 1992; and U.S. Patent Application No. 07 / 520,321, filed May 7, 1990. The targeting polynucleotide may include a sequence that facilitates the recombinase filling process, for example, a RecA filling sequence may be a recombinogenic nucleating sequence polynucleotide.
[d (AC)] and its complement poly [d (GT)]. Double-stranded sequence poly [d (A
-C) -d (GT) _n ] (n is 5 to 25) is a moderately repetitive element in the target DNA.

One single-stranded DN hybridized to a non-coiled DNA target
The RecA-protein mediated D-loop formed between the A (ssDNA) probes shows significant differences in stability when compared to those hybridized to relaxed or linear double-stranded DNA targets. The dsDNA target sequence located inside the relaxed linear DNA target hybridized by the ssDNA probe forms a single D-loop and is unstable after removal of the RecA protein (Adzuma, Genes Devel. 6:
1679 (1992); Hsieh et al, PNAS USA 89: 6492 (1992); Chiu et al., Biochemi
stry 32: 13146 (1993)). The probe DNA instability of this hybrid formed with a linear double stranded DNA target is most likely due to the incoming ssDNA probe WC base pairing with the complementary DNA strand of the double stranded target and the base in other DNA strands. Probably because of the confusion of pairing. The required high free energy in the unpaired ssDNA form in a protein-free single D-loop is due to the stored free energy inherent in the uncoiled DNA target or base pairing starting at the distal end of the bound DNA molecule. Either can be supplemented, allowing the exchange chains to entangle freely.

However, the addition of the second complementary ssDNA to the triplex-containing single D-loop stabilizes the protein-removed hybrid binding molecule by causing WC base pairing of the probe and the replacement target DNA strand. Become Second RecA-coated complementary ssDNA (
cssDNA) strand to a single D-loop containing three strands stabilizes protein-removing hybrid binding located away from the free ends of the double-stranded target DNA (Sen
a & Zarling, Nature Genetics 3: 365 (1993); Revet et al. J. Mol. Biol. 23
2: 779 (1993); Jayasena and Johnston, J. Mol. Bio. 230: 1015 (1993)). The resulting quadruplex structure, due to the analogy with the triplex single D-loop hybrid, has
-Named loop and has been shown to be stable in the absence of the RecA protein. This stability is such that restoration of WC base pairing in the parent duplex is due to two WC base pairings in the double D-loop (one WC base pair in each heteroduplex D-loop).
It seems to happen because it is necessary for the confusion of (C vs.). Reverse transition (double D-
Each base pairing in the loop (from the loop to the duplex) is not favored due to the energy of one WC base pair, and the pair of cssDNA probes thus dynamically binds to a double-stranded DNA target in a stable hybrid structure. Be captured. The stability of the double D-loop contact molecule in the internally located probe: target hybrid is an intermediate step before the progress of the homologous recombination reaction to the strand exchange phase. The double D-loop allows for the isolation of a stable multi-stranded DNA recombination intermediate.

In addition, when the targeting polynucleotide is used to produce an insertion or deletion in an endogenous nucleic acid sequence as described herein, the two complementary single-stranded targeting polynucleotides will have a PCT This allows the use of an internal homology clamp as described in the drawing of US98 / 05223. The use of internal homology clamps allows for the formation of stable, protein-depleted cssDNA: probe target hybrids with homologous DNA sequences that contain relatively small or large insertions and deletions within the homologous DNA target.
Without being bound by theory, these probes containing homologous insertions within the cssDNA probe: the target hybrids are cs
It appears to be stabilized by reannealing the sDNA probe and forming a novel DNA structure with an internal homology clamp. Similarly, within a linear DNA target (
Stable double D-loop hybrids formed at internal sites with homologous insertions (with respect to the cssDNA probe) are similarly stable. Since the cssDNA probe is kinetically captured within the dual target, the multi-stranded DNA intermediate of homologous DNA pairing is stable and promotes strand exchange.

In a preferred embodiment, the length of the internal homology clamp (ie, the length of the insertion or deletion) is about 1 to 50%, preferably about 1 to about 20%, of the total length of the targeting polynucleotide. , About 1 to about 10% are particularly preferred, but in some cases, the length of the deletion or insertion may be significantly greater. For domain homology clamps, complementarity within the internal homology clamp need not be perfect.

The polynucleotide for targeting used in the method of the present invention is typically derived from denaturation of a single-stranded nucleic acid, usually a DNA strand, or a double-stranded DNA, and is one (or both) of the target double-stranded nucleic acids. Complementary to the strand of Thus, a complementary single-stranded targeting polynucleotide is complementary to one strand of an endogenous target sequence (ie, Watson), while the other complementary single-stranded targeting polynucleotide is an endogenous target sequence. (Ie, click). The domain homology clamp sequence preferably includes at least 90-95% sequence homology with the target sequence to ensure sequence-specific targeting of the polynucleotide targeting the endogenous DNA domain target (but as outlined) Low sequence homology may also be tolerated). Each single-stranded target polynucleotide is typically about 50-6
Although 00 bases in length, short or long polynucleotides may also be used.

[0062] Once the gene family and domain sequence have been selected, the targeting polynucleotide is made as recognized in the art. For example, for large targeting polynucleotides, the plasmid may be manipulated to generate a gene sequence of appropriate size with a deletion or insertion in the gene of interest, and substantially corresponding or substantially corresponding to the endogenous target DNA sequence. Include at least one adjacent homology clamp that is complementary. Vectors containing the targeting polynucleotide sequences are typically grown in E. coli and isolated using standard molecular biology techniques. Alternatively, the targeting polynucleotides may require the formation of subfragments of the targeting polynucleotides, initially for particularly large targeting polynucleotides, typically followed by joining the subfragments to each other, typically by enzymatic ligation. Can be prepared in a single-stranded form by an oligonucleotide synthesis method.
In general, as will be appreciated by those skilled in the art, targeting polynucleotides may be chemically synthesized oligonucleotides, nick translated double-stranded DNA templates, polymerase chain reaction amplification (or ligase chain reaction amplification) of sequences, plasmids, phagemids, YAC,
Cosmids, bacteriophage DNA, other viral DNA or replication intermediates,
Or a purified restriction fragment thereof, as well as sequences of interest such as other sources of single or double stranded polynucleotides having the desired nucleotide sequence (eg, cloned cD
NA or a genomic clone, or a portion thereof).

When using the microinjection method, it may be preferable to use the transfection method with a linear sequence containing only the modified target gene sequence and no vector or selectable sequence. The modified gene site is determined by homologous recombination between the exogenous targeting polynucleotide and the endogenous DNA target sequence by careful primer selection and PCR, followed by the presence of a PCR product specific for the desired target event. (See Erlich et al., Incorporated herein by reference).
l., (1991) Science 252: 1643). Several experiments have already used PCR and have successfully identified and transfected the desired transfected cell line (Zimmer and Gruss, (1989), incorporated herein by reference). Nature 338: 1500
; Mouellic et al., (1990) Proc. Natl. Acad. Sci. USA 87: 4712; Shesely et.
al., (1991) Proc. Natl. Acad. Sci. USA 88: 4294). This approach is very effective when the number of cells that accept the exogenous targeting polynucleotide (i.e., by microinjection or with liposomes) is large, reducing the treated cell population to about 1 x
It is expanded to a cell group of 10 ⁴ cells (Capecchi, (1989) Science 244: 1288).
When the target gene is not present on the sex chromosome, or when the cell is derived from a female, both alleles of the gene can be targeted by sequential inactivation (Mortensen et al., (19
91) Proc. Natl. Acad. Sci. USA 88: 7036). Alternatively, animals heterologous to the target gene can be bred homologously as is known in the art.

The present invention allows for the introduction of modifications in a target nucleic acid containing a desired domain.
That is, the fact that the heterologous structure is tolerable in the targeting polynucleotide is twofold: first, the use of a heterologous domain homology clamp that can target genes encoding functional domains of a single protein or multiple proteins. (This results in various genotypes and phenotypes), and secondly, the insertion of modifications into the target sequence. Thus, typically, the targeting polynucleotide (or complementary polynucleotide pair) is a portion or region having a sequence that is not present in a preselected endogenous target sequence (i.e., a heterologous portion or mismatch). Having. The target sequence may be as small as one mismatched nucleotide or several mismatches, or may extend to about a few kilobases or more of the heterologous sequence.

Thus, in a preferred embodiment, the methods and compositions of the invention are used for inactivating a domain of a gene. That is, the exogenous targeting polynucleotide can be used to inactivate, reduce or alter the biological activity of one or more domains in a cell (or transgenic non-human animal or plant) gene. This finding is of particular use for the production of animal models of disease states, or for elucidation of gene function and activity similar to "knockout" experiments. Alternatively, reducing the biological activity of the wild-type gene,
Wild-type activity can be turned into a simulated disease state. This includes the genetic manipulation of non-coding gene sequences, including promoters, repressors, enhancers and transcriptional activation sequences, that affect gene transcription.

Thus, in a preferred embodiment, homologous recombination of the targeting polynucleotide and endogenous target sequence, for example, as a result of insertion of a PCR tag, presumably both within and outside the domain functional domain region An amino acid substitution of the endogenous target sequence,
Leads to an insertion or deletion. This generally results in the regulation or alteration of the gene function of the endogenous gene, including both reduction or elimination of function as well as promotion of function. The heterologous portion may be used to make insertions, deletions and / or substitutions in the intended endogenous target DNA sequence, and / or to make one or more nucleotide substitutions in the intended endogenous target DNA sequence. Then, the obtained recombination sequence (ie, the target recombination endogenous sequence) includes some or all sequence information of the heterologous portion of the targeting polynucleotide. Thus, the heterologous regions are used to create mutated sequences, ie, target sequence modifications. In this way, site-specific modifications can be made in different ways for different purposes.

The endogenous target sequence, which is generally a nucleic acid encoding a domain, can be perturbed in a variety of ways. As used herein, the term "perturb" includes alterations in the coding or non-coding sequence of an endogenous nucleic acid. In one preferred embodiment, the perturbed gene no longer produces a functional gene product. In another preferred embodiment, the perturbed gene produces a mutant gene product. In general, confusion can occur due to nucleotide substitutions, insertions, deletions or frameshifts.

[0068] In one embodiment, amino acids are substituted. This may result from insertion of a non-naturally occurring domain sequence into a domain target, or more specific changes to a particular sequence outside the domain sequence.

[0069] In one embodiment, the endogenous sequence is disrupted by the inserted sequence. As used herein, the term "insertion sequence" refers to one or more nucleotides inserted into an endogenous gene to cause confusion. Generally, the insertion sequence will be one as outlined herein.
It can be as small as a nucleotide or as long as a gene. As a non-gene insertion sequence, the sequence is at least one nucleotide, preferably about 1 to about 50 nucleotides, particularly preferably about 10 to about 25 nucleotides. The insertion sequence may include a polylinker sequence, with about 1 to about 50 nucleotides being preferred, and about 10 to 25 nucleotides being particularly preferred. The insertion sequence can be a PCR tag used to identify the first gene.

In a preferred embodiment, the inserted sequence can disrupt the endogenous gene and prevent its expression, as well as result in the expression of a novel gene product. Thus, in a preferred embodiment, the perturbation of the endogenous gene by the inserted sequence gene is performed by a method that allows transcription and translation of the inserted gene. The inserted sequence encoding the gene may be about 50 bp to 5000 bp cDNA or about 5000 bp to 500 bp.
It can range from 00 bp of genomic DNA. This can be done in various ways, as will be appreciated by those skilled in the art. In a preferred embodiment, the inserted gene targets the endogenous gene in such a way as to utilize an endogenous regulatory sequence, including a promoter, enhancer or regulatory sequence. In another embodiment, the insert sequence gene includes its own regulatory sequences, such as a promoter, enhancer or other regulatory sequence.

[0071] Particularly preferred insertion sequence genes include, but are not limited to, genes encoding a selection or reporter protein. In addition, the inserted sequence gene can be a modified or mutated gene.

The term “deletion” as used herein includes the removal of a portion of the nucleic acid sequence of an endogenous gene. Deletions range from about 1 to about 100 nucleotides, from about 1 to 5
Although 0 nucleotides are preferred, and about 1 to about 25 nucleotides are particularly preferred, in some cases the deletions may be longer and the removal of the complete domain functional domain, the complete endogenous gene and / or its regulatory sequences. Can be effectively included. Deletions can occur with a combination of substitutions or modifications, leading to the final modified endogenous gene.

In a preferred embodiment, the endogenous gene can be disrupted simultaneously by insertions and deletions. For example, some or all of the endogenous gene may be removed or replaced with an inserted sequence gene, with or without its regulatory sequences. In this way, for example, the sequences other than the regulatory sequence of the endogenous gene can be removed and replaced with the inserted sequence gene. The inserted sequence gene is under the control of an endogenous gene regulatory element.

The term “regulatory element” as used herein includes, but is not limited to, promoter sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, enhancer or activator sequences, dimerization sequences, and the like. Used to describe non-coding sequences that affect the transcription or translation of a gene. In a preferred embodiment, the regulatory sequences include a promoter and transcription start and stop sequences. Promoter sequences encode either constitutive or inducible promoters. The promoter can be a naturally occurring promoter or a hybrid promoter. Hybrid promoters, which are combinatorial elements of one or more promoters, are also known in the art and are useful in the present invention.

In addition to the domain homology clamp and the optional internal homology clamp, the targeting polynucleotides of the present invention may include additional components such as cellular uptake components, chemical substituents, purification tags and the like. May be included.

[0076] In a preferred embodiment, the at least one targeting polynucleotide comprises at least one cellular uptake component. As used herein, a "cell uptake component", when bound directly or indirectly to a targeting polynucleotide, is intracellular into at least one cell type of the targeting polynucleotide (eg, a hepatocyte). Means an agent that promotes uptake. The targeting polynucleotides of the present invention can be conjugated to the cellular uptake component, as desired, typically covalently or preferably non-covalently. Various methods of techniques for targeting DNA to specific cell types have been described. The targeting polynucleotides of the present invention can be conjugated to several cellular uptake components known per se in the art. Targeting polynucleotides for hepatocyte targeting are described in the literature and are hereby incorporated by reference (Wu GY and Wu CH (herein incorporated by reference). 1987) J. Biol. Chem. 262: 4429;
Wu GY and Wu CH (1988) Biochemistry 27: 887; Wu GY and Wu CH (1988) J. Bi
ol. Chem. 263: 14621; Wu GY and Wu CH (1992) J. Biol. Chem. 267: 12436; Wu
et al. (1991) J. Biol. Chem. 266: 14338; and Wilson et al. (1992) J. B.
iol. Chem. 267: 963; WO92 / 06180; WO92 / 05250; and W
O91 / 17761) asialoorosomucoid (ASOR) -poly-L-lysine conjugate.

Alternatively, the formation of the cellular uptake component is accomplished by incubating the targeting polynucleotide with at least one lipid species and at least one protein species, essentially comprising the targeting polynucleotide and the lipid-protein cell uptake component. To form a protein-lipid-polynucleotide complex. Felgner (
Other forms of lipid vesicles or polynucleotide applications prepared according to WO 91/17424, which is hereby incorporated by reference (EP 465,529, which is hereby incorporated by reference) may also be used as cell uptake components. Can be used. Nucleases can also be used.

In addition to a cellular uptake component, targeting components such as a nuclear localization signal may be used as is known in the art. For example, Kido, which is hereby incorporated by reference.
See, et al., Exper. Cell. Res. 198: 107-114 (1992).

Typically, a targeting polynucleotide of the invention is coated with at least one recombinase, conjugated to a cellular uptake component, and the resulting cell targeting complex is loaded under uptake conditions (eg, under physiological conditions), The target polynucleotide is contacted with the target polynucleotide and the recombinase is inwardly directed to the target cell. The targeting polynucleotide may be contacted simultaneously or sequentially with the cellular uptake component and also with the recombinase; preferably, the targeting polynucleotide is initially contacted with the recombinase, or with both the cellular uptake component and the recombinase, on average at least about So that one molecule of the recombinase binds non-covalently per target polynucleotide molecule,
At least about one cell uptake component is also contacted to non-covalently bind. Most preferably, the coating and cellular uptake components of both recombinases fill essentially all of the available binding sites for the targeting polynucleotide. The targeting polynucleotide is preferentially coated with the cellular uptake component such that the resulting targeting complex contains, on a molar basis, more cellular uptake component than the recombinase. Alternatively, the targeting polynucleotide is preferentially coated with the recombinase to include, on a molar basis, more recombinase than cellular uptake components.

A cellular uptake component is included with the recombinase-coated target polynucleotide of the present invention to enhance the uptake of the recombinase-coated target polynucleotide into cells. This enhancement is particularly useful in gene therapy to treat genetic disorders, including tissue overgrowth,
And targeted homologous recombination to treat viral infections. The viral sequence (eg, the integrated hepatitis B virus (HBV) genome or genomic fragment) is targeted and inactivated by the homologous sequence target. Alternatively, the target polynucleotide may be coated with a cellular uptake component and targeted to cells with simultaneous or simultaneous administration of the recombinase (e.g., a liposome or immunoliposome containing the recombinase, encoding and expressing the combinase). Virus-based vectors).

[0081] In addition to the recombinase and the cellular uptake component, at least one of the target polynucleotides may contain a chemical substituent. An exogenous target polynucleotide, modified with additional chemical substituents, is introduced into a metabolically active target cell along with a recombinase (e.g., RecA) and homologously paired with the intended endogenous DNA target sequence in the cell. can do. In a preferred embodiment, the exogenous target polynucleotide is derivatized, and additional chemical substituents are attached during or after polynucleotide synthesis, respectively, to direct specific endogenous target sequences that cause changes or chemical modifications to the local DNA sequence. Localize. Preferred additional chemical substituents include, but are not limited to, crosslinkers (Podyminogin et al., Biochem. 34; 13098 (1995) and 35).
: 7267 (1996), incorporated herein by reference), nucleic acid cleavage reagents, metal chelates (e.g., iron / EDTA chelates for iron-catalyzed cleavage), topoisomerases, endonucleases, exonucleases, ligases, Agents that normally bind to nucleic acids, such as phosphodiesterases, photochemical porphyrins, chemotherapeutic agents (e.g., adriamycin, doxylubicin, etc.), intercalating agents, labeling agents, base modifiers, labeling agents (e.g., Afonina et al., PNAS USA 93 : 3199 (1996), incorporated herein by reference), immunoglobulin chains and oligonucleotides. Iron / EDTA chelates are particularly preferred as the desired chemical substituent for local cleavage of DNA sequences (Hertzberg et al. (1982) J. Am. Chem. Soc. 104: 313; H
ertzberg and Dervan (1984) Biochemistry 23: 3934; Taylor et al. (1984) T
etrahedron 40: 457; Dervan, PB (1986) Science 232: 464, which is hereby incorporated by reference).

Further preferred is the hybridization of complementary single-stranded nucleic acids to one another.
(But not with unmodified nucleic acids) (e.g., Kutryav
in et al., Biochem. 35: 11170 (1996), and Woo et al., Nucleic Acid. Re.
s. 24 (13): 2470 (1996). Both are hereby incorporated by reference.)
2'-O methyl groups are also preferred (Cole-Strauss et al, Science 273: 1386 (1996
); Yoon et al., PNAS 93: 2071 (1996)). Additional preferred chemical substituents include label moieties including fluorescent labels. Preferred attachment chemistries include, for example, direct attachment via an attached reactive amino group (Corey and Schultz (19
86) Science 238 1401, incorporated herein by reference) and other direct binding chemistries, but also streptavidin / biotin and digoxigenin / anti-digoxigenin antibody binding methods can be used. Binding chemical substituents are disclosed in U.S. Patents 5,135,720, 5,093,245 and 5,055,556, which are hereby incorporated by reference. Other binding chemicals can also be used at the discretion of the skilled practitioner.

[0083] In a preferred embodiment, at least one target polynucleotide comprises at least one purification tag or catcher moiety. Some of them have been described above as chemical substituents, for example, biotin, digoxigenin, psoralen and the like. Alternatively, domain oligonucleotides can be directly attached to beads in a target reaction performed on a solid support.

In a preferred embodiment, the target polynucleotide is coated with a recombinase prior to the introduction of the domain target. Conditions for coating a target polynucleotide with a recombinase such as RecA protein and ATPγS are described in US patent application Ser. No. 07 / 910,791 filed on Jul. 9, 1992; And U.S. Patent Application Serial No. 07 / 520,321 filed May 7, 1990 and PCT US98 / 05223, incorporated herein by reference. Although E. coli RecA is used directly in the following manner, other recombinases can be used as well, as will be apparent to those skilled in the art. GTPγS, a mixture of ATPγS and rATP, rGTP and / or dATP, or dATP or r in the presence of a rATP generation mechanism
ATP alone can be used to coat a target polynucleotide (Boehringe
r Mannheim). Various mixtures such as GTPγS, ATPγS, ATP, ADP, dATP and / or rATP or other nucleosides can be used,
Particularly preferred is a mixture of ATPγS and ATP or ATPγS and ADP.

The RecA protein coating of the target polynucleotide is described in US patent application Ser. No. 07 / 910,791 filed Jul. 9, 1992 and US patent application Ser. It is usually done in the manner described in PCT US98 / 05223 (hereby incorporated by reference). Briefly, either the double-stranded or single-stranded target polynucleotide is denatured by heating in aqueous solution at 95-100 ° C for 5 minutes. Then in an ice bath for 20 seconds to about 1
Centrifuge at 0 ° C. for about 20 seconds before use. If the denatured target polynucleotide is not placed in a −20 ° C. freezer, it is usually immediately added to a standard RecA coating reaction buffer containing ATPγS at room temperature. To this is added the RecA protein. Alternatively, the RecA protein may be included with the buffer component and ATPγS before adding the polynucleotide.

RecA coating of the target polynucleotide is initiated by incubation with the polynucleotide-RecA mixture at 37 ° C. for 10-15 minutes. The RecA protein concentration examined during the reaction with the polynucleotide is the size of the polynucleotide and the amount of polynucleotide added, and the RecA molecule: nucleotide ratio
(Preferably in the range of about 3: 1 to 1: 3). When the single-stranded polynucleotide is RecA-coated independent of the homologous polynucleotide chain, the mM and μM concentrations of ATPγS and RecA, respectively, are reduced by a factor of two when using a double-stranded target polynucleotide. (Ie, RecA and A
The TPγS concentration ratio is usually kept constant at a particular concentration of the individual polynucleotide strand, depending on whether single or double polynucleotide is used).

The RecA protein coating of the target polynucleotide is typically a standard 1 × Re
Performed in a cA coating reaction buffer. 10x RecA reaction buffer (ie, 10
× AC buffer) was 100 mM Tris acetate (pH 7.5, 37 ° C.), 20 mM
Consists of magnesium acetate, 500 mM sodium acetate, 10 mM DTT and 50% glycerol. All target polynucleotides, either single-stranded or double-stranded, are heated at 95-100 ° C. for 5 minutes before use, placed in an ice bath for 1 minute, and then placed at 0 ° C.
Centrifugation (10,000 rpm) for about 20 seconds (eg, a Torny centrifuge) to generally denature. Normally denatured target polynucleotide is quickly added to room temperature RecA coating reaction buffer mixed with ATPγS and diluted with twice the volume of H ₂ O if necessary.

The reaction mixture usually contains the following components: (i) 0.2-4.8 mM ATPγS and (ii) a target polynucleotide in the range of 1-100 ng / μl. About 1-
20 μl RecA protein (for 10-100 μl reaction mixture) is added and usually about 2-10 mg / ml (purchased or purified from Pharmacia) is quickly added and mixed. The final reaction volume for RecA coating of the target polynucleotide is typically in the range of about 10-500 μl. The RecA coating of the target polynucleotide is usually
It is started by incubation at 37 ° C. for 10-15 minutes with the target polynucleotide-RecA mixture.

The RecA protein concentration varies depending on the size of the target polynucleotide and the amount of the target polynucleotide added (RecA protein concentration usually ranges from 5 to 50 μM). If the single-stranded target polynucleotide is RecA coated without dependence on the homologous polynucleotide chain, the concentration of ATPγS and RecA proteins will be about 1% when using a double-stranded target polynucleotide.
/ 2. That is, the RecA protein and ATPγS concentration ratios are:
Usually, the individual polynucleotide chains are kept constant at a certain concentration.

[0090] Coating of the target polynucleotide with the RecA protein can be assessed in a variety of ways. First, protein binding to DNA can be tested using a band shift gel assay (McEntee et al., (1981) J. Biol. C
hem. 256: 8835). The labeled polynucleotide can be coated with the RecA protein in the presence of ATPγS, and the products of the coating reaction can be separated by agarose gel electrophoresis. Following incubation of the RecA protein with the denatured double-stranded DNA, the RecA protein effectively coats the single-stranded target polynucleotide derived from denaturation of the double-stranded DNA. The ratio of RecA protein monomer to nucleic acid in the target polynucleotide is 0, 1:27 for the 121-mer.
From 1: 2.7 to 3.7: 1, 0 for the 159 mer, 1:22, 1: 2.2 to 4.
As the ratio increased to 5: 1, the electrophoretic mobility of the target polynucleotide decreased. That is, the delay is due to RecA binding to the target polynucleotide. The mobility of the coated polynucleotide reflects the saturation of the target polynucleotide with the RecA protein. The excess of RecA monomer to DNA nucleotides requires effective RecA for short target polynucleotides (Leahy et al., (1986) J. Biol Chern. 261: 954).

A second method for assessing protein binding to DNA is to use a nitrocellulose filter binding assay (Leahy et al, (1986) J. Biol Chem. 261: 6).
954; Woodbury, et al., (1933) Biochemistry 22 (22): 4370-4373). This nitrocellulose filter binding method uses a protein using labeled DNA: DN.
It is particularly useful for measuring the dissociation ratio of the A complex. In the filter binding method, DNA
: The protein complex is retained on the filter, while free DNA passes through the filter. This assay method has good quantitative properties as a measurement of the dissociation rate. Purification of the DNA: protein complex from the free target polynucleotide can be very rapid.

Alternatively, the recombinase protein (prokaryotic, eukaryotic or exogenous to the target cell) can be administered to the target cell simultaneously or simultaneously with the target polynucleotide (
Exogenous induction or treatment (ie, within about a few hours) may be used. These treatments are usually performed by macroinjection, but electroporation lipofection and other transfection methods well known to those skilled in the art may be used. Alternatively, the recombinase-protein may be prepared in vivo. For example, it may be prepared from a homologous or heterologous expression cassette in transfected or target cells. For example, a transgenic totipotent cell (eg, a fertilized zygote) or an embryonic stem cell (eg, a mouse ES cell such as AB-1) is a transgenic non-human that reconstitutes all or a portion of an individual stem cell individual (eg, a hematopoietic). Used to produce human animal strains or somatic cells or pluripotent hematopoietic stem cells.
Advantageously, heterologous expression cassettes include ecdysone-inducible promoter-enhancer complex, estrogen-inducible promoter-enhancer complex, CM
Other cell type-specific, developmental stage-specific, hormone-induced drug derivatives, such as regulatable promoters, such as the V promoter-enhancer, the insulin gene promoter, or tetra or other regulatable promoter constructs, are included.
The expression of at least one recombinase protein from the cassette is regulated to produce a transient recombinase in vivo concurrently or concurrently with the injection of the target polynucleotide into the cell. When using a hormone-inducible promoter-enhancer complex, the cells must have the required hormone receptor presentation (either native or the result of expressing a transfected expression vector encoding these receptors). Alternatively, the recombinase may be endogenous and prepared at high levels. In this embodiment, preferably a eukaryotic target cell such as a tumor cell, wherein the target cell prepares an enhanced level of recombinase. In another embodiment, the level of recombinase may be derived from a DNA damaging agent such as mitomycin C, UV or gamma irradiation. Alternatively, a plasmid encoding the recombinase gene in the cell can be used to increase recombinase levels.

It has been found that the compositions of the present invention, once made, can be used in a variety of applications for treating target cells. In general, the compositions and methods of the invention are useful for identifying new members of the gene family that are useful in functional genomic studies as well as for the identification of new pharmaceutical targets. Both of these can be done through the generation of a "knockout" animal model. In addition, the present invention can be used to modify functional domain targets, generate transgenic plants and animals, clone genes containing domain functional domains, and the like.

[0094] By producing and administering to a target host cell, it is determined that the compositions of the present invention can be used for many applications, including domain-specific gene deployment. A polypeptide or protein encoded by a target nucleic acid undergoes homologous recombination with a plurality of polynucleotides to yield a number of modified target nucleic acids, which are expressed to yield a number of modified proteins. A selection system is employed to identify and isolate host cells that express a protein having the desired properties or phenotype. For example, if the expressed protein is an enzyme, cells with modified enzymatic activity are identified. For a desired activity, the activity may be increased or decreased or modified. A protein having the desired phenotype is selected and isolated, the modified nucleic acid is sequenced to identify a sequence that provides the desired activity, and the process is repeated as necessary to obtain a protein having the desired activity or property. Produce.

In this and other embodiments, a preferred target sequence is a therapeutically or commercially suitable protein, such as, but not limited to, an enzyme (
Protease, recombinase, lipase, kinase, carbohydrase, isomerase, tautomerase, nuclease, etc.), hormones, receptors, transcription factors, growth factors, cytokines, globin genes, immunosuppressive genes, tumor suppressors, oncogenes, complement activating genes, Milk proteins (casein, α-lactalbumin, β-lactalbumin, bovine and human serum albumin), immunoglobulins, milk proteins, pharmaceutical proteins and vaccines.

In a preferred embodiment, the method of the invention is used to generate a pool or library of variant nucleic acid sequences and a library of cells containing the variant sequences. This concept is somewhat similar to "gene shuffling" in the literature (Stemmer et al., 1994,
(See Nature 370: 389; this document attempts to "spread" genes rapidly by making multiple random changes simultaneously.) In the present invention, this object is achieved by using at least one cycle, preferably a repetitive cycle, of increased homologous recombination with the targeting polynucleotide, including random mismatches, substitutions, insertions, and deletions. By using a library of target polynucleotides containing many random mutations and repeating homologous recombination as many times as necessary, rapid “gene expansion” occurs, in which new genes are replaced by a large number of mutations Will be included.

Thus, in this embodiment, a number of targeting polynucleotides are used. Each targeting polynucleotide has at least one homologous clamp that substantially corresponds to or is substantially complementary to the target sequence. Generally, targeting polynucleotides are produced in pairs; that is, two single-stranded targeting polynucleotide pairs that are substantially complementary to each other are prepared (ie, a Watson strand and a Crick strand). However, as the skilled artisan will appreciate, less than a 1: 1 Watson-Crick strand may be used; for example, a single-stranded targeting polynucleotide (ie, a Watson strand).
Can be used in excess. Preferably, a sufficient number of each of the Watson and Crick strands is used so that the majority of the targeting polynucleotides can form a double-stranded D-loop, which is a single-stranded D-loop as described above. More preferred. Further, the pair need not be perfect in complementarity; for example, if one of the single-stranded target polynucleotides (ie, the Watson strand) (which may or may not contain a mismatch) is in excess. Can pair with a very large number of mutant click chains, and so on.
Due to the randomness of the pairing, one or both specific pairs of single-stranded targeting polynucleotides do not contain mismatches. However, generally at least one strand contains at least one mismatch.

[0098] The plurality of pairs preferably comprises a mismatched pool or library. The size of a library depends on many factors, such as the number of residues to be mutagenized, the sensitivity of the protein to mutations, etc., as will be appreciated by those skilled in the art. In general, the library in this case will preferably contain at least 10% different mismatches over the entire length of the targeting polynucleotide, with at least 30% being preferred, especially at least 40% being preferred, but As will be appreciated, in some cases lower (1, 2, 5%, etc.) or higher amounts of mismatches are possible and desirable. That is, the plurality of pairs comprises a random pool and preferably generate mismatches over some or all regions of the entire targeting sequence. As outlined herein, "mismatch" includes substitutions, insertions, and deletions, with the former being preferred. Thus, for example, a pool of degenerate variant targeting polynucleotides that span some, or preferably all, possible mismatches for a region is generated using techniques well known in the art, as described above. . Preferably, but not necessarily, the variant targeting polynucleotide contains one or a few mismatches (10 or fewer) each, allowing full multiple randomization. That is, by repeating the homologous recombination step as many times as possible, mismatches from many probes can be incorporated into the single-stranded target sequence, as described in more detail below.

The mismatch may be non-random (ie, targeted) or random, and may be biased random. That is, in some cases, a specific change is desirable, and thus the sequence of the target polynucleotide is specifically selected. In a preferred embodiment, the mismatch is random. The targeting polynucleotide can be chemically synthesized, thus incorporating the nucleotide at any position. Synthetic methods can be designed to produce randomized nucleic acids, allowing the formation of all or most of the possible combinations over the entire length of the nucleic acid, and forming a library of polynucleotides for randomized targeting. Preferred methods maximize the size and diversity of the library.

It is important to understand that in a library system encoded by oligonucleotide synthesis, there may not be complete control over the codons that will eventually be incorporated into the peptide structure. This means that the codon is a stop signal (TA
A, TGA, TAG). In the synthesis with NNN as a random region, the probability that the codon is a stop codon is 3/64, or 4.69%. To alleviate this, random residues are coded as NNK (where K = T or G). This allows the encoding of all possible amino acids (slightly altering their relative expression levels), but what is important is to prevent the encoding of the two stop residues TAA and TGA. .

In one embodiment, the mismatches are sufficiently randomized that there is no sequence preference or constantity at any position. In a preferred embodiment, the library is skewed. That is, certain parts of the sequence are kept constant or are selected from a limited number of possibilities. For example, in a preferred embodiment, nucleotides or amino acid residues are randomized within a particular class, such as hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues. Cysteine for cross-linking, proline for SH-3 domains, serine for phosphorylation sites, threonine, tyrosine, histidine, etc., or for purines.

As will be appreciated by those skilled in the art, introducing a pool of variant targeting polynucleotides (in combination with a recombinase) into a target sequence, ie, an extra-chromosomal sequence in vitro, results in a tremendous amount of time over time. Results in a high number of homologous recombination reactions. That is, a significant number of homologous recombination reactions can occur on a single-stranded target sequence, resulting in a variety of single and multiple mismatches within the single-stranded target sequence and a library of such variant target sequences. However, most of them contain mismatches and are different from other elements of the library. Thus, this acts to generate a mismatched library.

In a preferred embodiment, the variant targeting polynucleotide is made to a particular region or domain of the sequence (ie, a nucleotide sequence encoding a particular protein domain). For example, it is desirable to generate a library of all possible variant forms of a protein binding domain without affecting different biologically functional domains and the like. Thus, the methods of the present invention find particular use in generating a number of different variants within a particular region of a sequence, as well as cassette mutagenesis that is not limited by sequence length. This concept is sometimes referred to herein as "domain-specific gene expansion." In addition, more than one region can be changed simultaneously by these techniques; thus, "single domain" and "multiple domain" shuffling can be performed. Suitable domains include, but are not limited to, kinase domains, nucleotide binding sites, DNA
Binding site, signal transduction domain, receptor binding domain, transcription activation region,
Promoter, origin of replication, leader sequence, terminator, localization signal domain, complementarity-determining regions (CDRs) in immunoglobulin genes, Fc, _VH and _VL .

In a preferred embodiment, a variant targeting polynucleotide is generated for the entire target sequence. In this way, the majority of single and multiple mismatches can be made for the entire sequence.

Thus, the present embodiment proceeds as follows, as generally shown in FIGS. A pool of targeting polynucleotides is created such that each contains one or more mismatches (referred to as "combinatorial domain-specific gene deployment probes" in the figure). The probe is coated with the recombinase described generally herein and introduced into the target sequence. Based on the binding of the probe forming the D loop,
The recombinase is preferably removed. These polynucleotide: target sequences can then be introduced into recombinantly operable cells to produce the target protein, which can be tested for biological activity based on the identification of the target sequence. Depending on the results, the repeated target sequence can be used as a starting target sequence by repeating homologous recombination, but generally the same library is used. In a preferred embodiment, at least two homologous recombinations are used, but preferably at least 5 times, particularly preferably at least 10 times. Again, the number of iterations performed depends on the desired end point, the resistance or susceptibility to protein mutation, the mismatch in each probe, and the like.

In a preferred embodiment, the target sequence is an immunoglobulin. The amino-terminal regions of the antibody light and heavy chains together form the antigen-binding site and the variable region of their amino acid sequences, but that region forms the structural basis for antigen-binding site diversity. provide. The variability of both heavy and light chain variable regions is largely limited to three small hypervariable regions in each chain. The rest of the variable region, known as the framework region, is relatively constant. Hypervariable regions each consist of only 5 to 10 amino acids; the corresponding regions of the DNA encoding these regions are known as complementarity determining regions or CDRs. In this way, the sequences of both heavy and light chain CDR regions can be altered to create an antibody library, for example, an antibody phage library. Different permutation combinations of CDRs can be changed and deployed in an antibody phage library.

[0107] In a preferred embodiment, the target sequence is a single-chain Fv framework for a number of specific antigens. Single-chain Fv (scFv) consists of the _VL and _VH domains of an immunoglobulin linked by a peptide spacer and thus contains the minimal antigen-binding domain of an antibody.

[0108] In a preferred embodiment, an antibody-phage fusion is used as the target sequence.
As is known in the art, a single-chain Fv fusion with a pIII minor coat protein allows expression of the antibody on the surface of the phage, where it can be used for antigen binding. Five copies of pIII are expressed on the phage surface. Therefore, it is possible to express five scFv on phage. This antibody-phage display system has already been used to isolate new antibodies. Starting with antibodies to any antigen, high affinity antibodies can be made as well as new antibodies.

In a preferred embodiment, the target sequence is a coding sequence for β-lactamase. Thus, the method of the present invention provides, for example, lacZ and green fluorescent protein (GF
Higher recombinant reporter genes such as P); higher antibiotic drug resistance genes; higher recombinase genes; higher recombinant vectors; and other higher recombinant genes, and for example, immunoglobulins, vaccines or therapeutics Can be used to create other proteins that have the value of For example, targeting polynucleotides containing a significant number of modifications can be made to one or more functional or structural domains of the protein and the products of homologous recombination can be evaluated.

By preparing a target cell and administering it to the cell, the target cell can be selected and a cell containing the modification of the target sequence can be identified. This can be done in a number of ways, but as will be recognized by those skilled in the art, will depend on the target gene and the targeting polynucleotide. The selection depends on the target sequence, its phenotypic, biochemical, genotypic,
Or it may be based on other functional changes. In further embodiments, as will be appreciated by those skilled in the art, selectable markers or marker sequences may be included in the targeting polynucleotide to facilitate subsequent identification.

In a preferred embodiment, there is provided a kit comprising a composition of the invention. The kit includes the composition, particularly a composition comprising a library or pool of denatured cssDNA probes, along with a number of reagents such as recombinases, buffers, ATP, and the like.

In an alternative embodiment, the targeting polynucleotide: target nucleic acid complex acts as a substrate for single-stranded endonucleases such as S1 and P. nuclease. Preferably, the targeting polynucleotide is substantially complementary and forms a double-stranded D loop with the target nucleic acid. The conjugation of the complex is single-stranded in nature and is therefore sensitive to single-strand-specific and conjugate-specific nucleases. Therefore,
Treatment of the complex with a single-stranded nuclease results in a well-defined nick in a selected region that encodes the intended domain of the protein encoded by the target nucleic acid. The cut target nucleic acid is dissociated from the target polynucleotide, assembled and shuffled by in vitro PCR (Stemmer, 1994, Nature 370: 389-391) to produce a plurality of modified nucleic acids. The modified nucleic acid is introduced into a suitable host cell for expression of a plurality of modified proteins as described above. Cells that express the modified protein are identified and isolated using selection techniques as described herein. This step is repeated as necessary to further develop the targeted nucleic acid.

In a preferred embodiment, the use of the present invention involves the isolation of new members of the gene family. As shown generally in FIG. 1, the use of a domain filament (ie, a domain homology clamp that preferably contains one purification method, such as the use of a purification tag such as biotin, disoxysenin or a RecA antibody) results in a gene family. New genes within are identified. Once identified, the new gene
It can be cloned and sequenced, and the protein gene product is purified. As will be appreciated by those skilled in the art, the functional significance of novel genes is assessed in a number of ways, including functional studies on protein levels and the generation of knockout animal models. By selecting therapeutically relevant gene family domain sequences, novel targets that can be used for drug candidate screening can be identified.

Thus, in a preferred embodiment, the present invention provides a method for isolating novel members of a gene family. The method comprises providing a targeting polynucleotide comprising a domain homology clamp and at least one purification tag, preferably biotin,
For example, introduced into a nucleic acid mixture such as a plasmid cDNA library or cells,
Then, utilizing the purification tag to isolate the gene. The exact method depends on the purification tag: the preferred method utilizes the attachment of the tag's binding ligand to the beads and is used to retrieve the sequence. Alternatively, anti-RecA antibodies can be used to capture RecA-coated probes. The gene is then cloned, sequenced and reassembled, if necessary, in a manner known to those skilled in the art.

In another preferred embodiment, the use of the invention has use in functional genetic research, by providing for the production of disease models in transgenic animals. Thus, for example, HMT used in homologous recombination methods can produce animals with very diverse mutations in a wide variety of related genes and produce a very diverse phenotype, including phenotypes related to disease states. Potentially bring. That is, by targeting a gene family, one, two or many genes in the family are modified in any particular experiment to create a very diversely assessed genotype and phenotype. Thus, in a preferred embodiment, the compositions and methods of the invention are used to create a pool or library of variant nucleic acid sequences. In the pool or library, the mutations are in the domain encoding domain, functional domain, cell library including variant library and animal library including variant library.

In addition, domain targeting may be used in diseased or altered cells or animals; in essence, domain targeting is a “reverting” gene, either within the same gene family or a completely different gene family. To identify genes that can regulate disease states caused by different genes. Therefore,
For example, loss of one type of enzyme activity results in a disease phenotype, complemented by alterations in different but homologous enzyme activities.

Thus, once a polynucleotide composition for recombinase targeting is produced, the composition is introduced or administered to target cells. Administration is typically performed as is known for administering nucleic acids to cells, and as the skilled artisan will appreciate, the method depends on the choice of target cells. Suitable methods include, but are not limited to, microinjection, electroporation,
Lipofection and the like. As used herein, "target cell" refers to a prokaryotic or eukaryotic cell. Suitable prokaryotic cells include, but are not limited to, bacteria such as E. coli Bacillus species and extremophile bacteria such as thermophilic fungi and halophilic fungi.
Preferably, the prokaryotic target cells are recombinantly competent. Suitable eukaryotic cells include, but are not limited to, fungi such as yeast and filamentous fungi, including species of the genus Aspergillus, Trichoderma, and Neurospora; corn, sorghum, tobacco, canola Plant cells such as soybeans, cotton, tomatoes, potatoes, alfalfa, sunflowers; and animal cells such as fish, reptiles, amphibians, birds and mammals. Suitable fish cells include, but are not limited to, cells from the species salmon, trout, tilapia, tuna, carp, flounder, halibut, swordfish, cod and zebrafish. Suitable avian cells include, but are not limited to,
Includes cells such as chickens, ducks, quails, pheasants, ostriches, turkeys, and other wild birds or game birds. Suitable mammalian cells include, but are not limited to, horses, cows,
Rodents such as buffalo, deer, sheep, rabbits, mice, rats, hamsters and guinea pigs; marine mammals such as goats, pigs, primates, dolphins and whales; cells of any tissue or stem cell type human cell line Lines, and stem cells, including pluripotent and non-pluripotent, and non-human zygotes. Specific human cells include, but are not limited to, all types of tumor cells, especially melanoma, myeloid leukemia, and lung, breast,
Ovarian, colon, kidney, prostate, pancreatic and testicular tumors), cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T-cells and B cells), mast cells, eosinophils, endothelial cells,
Stem cells such as hepatocytes, leukocytes including mononuclear leukocytes, hematopoietic, neural, skin, lung, kidney, liver and muscle cells, osteoclasts, chondrocytes and other related tissue cells, keratinocytes, melanocytes , Liver cells, kidney cells and fat cells. Suitable cells also include, but are not limited to, Jurkat T cells, mouse La, HT1080,
Known research cells including C127, Rat2, CV-1, NIH3T3 cells, CHO, COS, 293 cells and the like are included. See ATCC cell line catalog (herein incorporated by reference).

[0118] In a preferred embodiment, the prokaryotic cell identifies a gene family member,
Used to clone or alter. In this embodiment, the preselected target DNA sequence is selected for alteration. Preferably, the preselected target DNA sequence is contained within an extrachromosomal sequence. As used herein, “extrachromosomal sequence” refers to a sequence separated from a chromosomal or gene sequence. Preferred extrachromosomal sequences include plasmids (particularly prokaryotic plasmids such as bacterial plasmids).
, P1 vector, viral genome, yeast, bacterial and mammalian artificial chromosomes (YAC, BAC and MAC, respectively) and, although not required, other self-replicating sequences. As described herein, a recombinase and at least two
The strand targeting polynucleotides are substantially complementary to each other and each contain a homology clamp to a target sequence contained on the extrachromosomal sequence, preferably added to the extrachromosomal sequence in vitro. The two single-stranded targeting polynucleotides are:
Preferably, it is coated with a recombinase and at least one of the targeting polynucleotides contains at least one nucleotide substitution, insertion or deletion. The targeting polynucleotide then binds to the target sequence in the extrachromosomal sequence, resulting in homologous recombination, forming a modified extrachromosomal sequence containing substitutions, insertions or deletions. The altered extrachromosomal sequence is then introduced into prokaryotic cells using techniques known to those skilled in the art. Preferably, the recombinase is removed using techniques known to those skilled in the art before introduction into the target cells. For example, proteases such as proteinase K, SD
Treat the reaction with a detergent such as S and a phenol extraction method (including phenol: chloroform: isoamyl alcohol extraction method). These methods can also be used on eukaryotic cells. Cell growth, allowing the expression of variant nucleic acids, variant proteins,
In particular, it is performed under conditions that form a protein having a modification in the domain.

In a preferred embodiment, proteins having the desired phenotype are selected and isolated, and the sequence of the modified nucleic acid is determined to identify sequences that exert the desired activity. This process is repeated as necessary to produce a protein having the desired activity or properties.
Thus, in a preferred embodiment, the method of the invention is repeated until the desired protein or phenotype is found.

[0120] Alternatively, the preselected target DNA sequence is a chromosomal sequence. In this embodiment, the recombinase along with the targeting polynucleotide is introduced into a target cell, preferably a eukaryotic target cell. In this embodiment, it may be desirable to couple (generally non-covalently) the nuclear localization signal to the targeting polynucleotide to facilitate localization of the complex in the nucleus. See, for example, Kido et al., Exper
Cell Res. 198: 107-114 (1992), which is hereby incorporated by reference. The targeting polynucleotide and the recombinase result in homologous recombination, resulting in an altered chromosomal or gene sequence.

In a preferred embodiment, eukaryotic cells can be used. Basically, mammalian cells are used. Mouse, rat, primate and human cells are particularly preferred. And
Suitable cell types include, but are not limited to, all types of tumor cells, such as fibroblasts, epithelial cells (especially melanoma, bone marrow leukocytes, lung, breast, ovary, colon, kidney, prostate, pancreas, Testicular carcinoma), cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T cells and B cells), mast cells, neutrophils, circulating intimal cells, hepatocytes, leukocytes including mononuclear leukocytes, hematopoiesis, nerves, Stem cells such as skin, kidney, liver and muscle (used to select differentiation and undifferentiated factors), osteoclasts, connective tissue cells such as chondrocytes, keratinocytes, black cells,
Hepatocytes, kidney cells, and fat cells. Suitable cells include known research cells, including but not limited to Jurkat T cells, NIH3T3 cells, CHO, Cos, and the like. See, AYCC cell line catalog (herein incorporated by reference).

To produce transgenic non-human animals, including homologously targeted non-human animals, embryonic stem cells (ES cells), donor cells for nuclear transfer and fertilized zygotes are preferred. In a preferred embodiment, embryonic stem cells are used. As described (Robertson, EJ (1987) in Teratocarcinomas and Embryonic Stem Cells
: A Practical Approach. EJ Robertson, ed. (Oxford: IRL Press), p.71-11
2; Zjilstra et al., Nature 342: 435-438 (1989); and Schwartzberg et al., S
cience 246: 799-803 (1989), each of which is incorporated herein by reference), the AB-1 line (M
Mouse ES cells such as cMahon and Bradley, Cell 62: 1073-1085 (1990)) can be used for homologous gene targeting. Other suitable ES systems include, but are not limited to,
E14 strain (Hooper et al. (1987) Nature 326: 292-295), D3 strain (Doetschman et al.
al. (1985) J. Embryol. Exp. Morph. 87: 21-45) and the CCE system (Robertson et al.
al. (1986) Nature 323: 445-448). Whether a mouse line can be produced from ES cells with targeted specific mutations depends on the pluripotency of the ES cells (ie, once injected into the host blastocyst, participates in embryogenesis and transforms the germ cells of the resulting animal). Ability to bring)
Depends on.

The pluripotency of any particular ES cell line can vary depending on the time in culture and the care taken when processing. The only assay that can be relied on for pluripotency is to determine whether the specific population of ES cells used for targeting can give rise to chimeras that can harbor the germline of the ES genome. For this reason, prior to gene targeting, a portion of the parental population of AB-1 cells could be injected into C57B1 / 6J blastula to determine whether the cells could produce chimeric mice with excess ES cell donation, and whether Determine if most of the chimeras can transfer the ES genome to progeny.

In a preferred embodiment, non-human zygotes are used, for example, to create transgenic animals using techniques known to those of skill in the art (see US Pat. No. 4,873,1).
No. 91; Brinster et al., PNAS 86: 7007 (1989); Susulic et al., J. Biol. Ch.
em. 49: 29483 (1995), and Cavard et al., Nucleic Acids Res. 16: 2099 (1988)
, Incorporated herein by reference). Preferred zygotes include animal zygotes, including but not limited to fish, birds, reptiles, amphibians and mammalian zygotes.
Suitable fish zygotes include, but are not limited to, zygotes from species such as salmon, trout, tuna, carp, flounder, halibut, swordfish, cod, tilapia and zebrafish. Suitable avian zygotes include, but are not limited to, chicken, duck, quail,
Includes conjugates of pheasants, turkeys, and other wild chickens or game birds. Suitable mammalian zygotes include, but are not limited to, horses, cows, buffalo, deer, sheep, rabbits, mice,
Includes cells from rodents such as rats, hamsters and guinea pigs, marine mammals such as goats, pigs, primates, dolphins, whales and the like. See Hogan et al.,
Manipulating the Mouse Embryo (A Laboratory Manual), 2nd Ed. Cold Spring
Harbor Press, 1994 (incorporated herein by reference).

The vector containing the DNA segment in question can be transferred to a host cell by known methods, depending on the type of cellular host. For example, calcium phosphate treatment, electroporation, lipofection, biolistic or virus-based transfection may be used, but microinjections are typically used for target cells. Other methods used to transform mammalian cells include the use of polybrene, prototypic fusion, and the like (see, generally, Sambrook et al. Molecular Cloning: A Laboratory
Manual, 2d ed., 1989, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, incorporated herein by reference). Direct injection of DNA and / or recombinase-coated targeting polynucleotides into target cells, eg, skeletal or muscle cells, may also be used (Wolff et al. (1990) Scien
ce 247: 1465, incorporated herein by reference.)

In a preferred embodiment, the progenitor animal or progenitor cell already contains a disease allele. As used herein, the term “disease allele” refers to an allele of a gene that can produce a recognizable disease. Disease alleles are dominant or recessive and can cause disease, either directly or when present with a specific genetic background or pre-existing pathological condition. Disease alleles may be present in the gene pool or may be newly produced in an individual by somatic mutation. For example, without limitation, disease alleles include: activating oncogene, sickle cell anemia allele, Tay-Sachs allele, cystic fibrosis allele, Resh-Nyhan allele, retinoblastoma susceptibility There are alleles, Fabry disease alleles, Huntington's disease allele, and xenoderma pigmentosa allele. As used herein, disease alleles include both alleles for human diseases and alleles for cognitive veterinary diseases. For example, the ΔF508 CFTR allele in human disease alleles associated with cystic fibrosis in North Americans.

Once produced and administered to target cells, new members of the gene family can be isolated as described herein.

[0128] Alternatively, target cells are screened to identify cells that contain functional domain sequence modifications of the targeted domain. This can be done in many ways, as will be appreciated by those skilled in the art, depending on the target gene and the targeting polynucleotide. Screening is based on phenotype, biochemical, genotype or other functional change and is dependent on the target sequence. For example, IgE levels are assessed for inflammation or asthma; vascular tone or blood pressure is assessed for hypertension, behavioral screening is not for neurological effects, lipoprotein profiles are screened for cardiovascular effects; secreted molecules are for endocrine methods. Evaluate; CBC evaluates for hematology studies. In further embodiments, selectable markers and marker sequences are included in the targeting polynucleotide to facilitate later identification, as will be appreciated by those skilled in the art.

In a preferred embodiment, there is provided a kit comprising the composition of the invention. The kit includes the composition, particularly a library or pool of denatured cssDNA probes, along with a number of reagents or buffers, including recombinases, buffers, salts, ATP, and the like.

The broad scope of the present invention can be best understood by reference to the following examples. The following examples do not limit the invention in any way. All patents, patent applications, and published references cited herein are hereby incorporated by reference.

Examples: Example 1 Development of β-lactamase domain-specific genes The nucleoprotein filament probes used in this study consisted of a small DNA oligonucleotide library of 20-200 base pairs coated with RecA protein. Oligonucleotide libraries contain regions of random sequence flanked by homology clamps that hybridize to a β-lactamase domain that catalyzes the cleavage of the β-lactam ring of ampicillin. Oligonucleotide libraries are synthesized by standard solid phase methods and purified by electrophoresis on a 6% denaturing polyacrylamide gel. The probes are end-labeled by standard ^{32 P} methods and / or biotin. The addition of single-stranded DNA to the RecA protein forms a nucleoprotein filament (at a ratio of 1 RecA molecule to 3 nucleotides). This method is described in Sena and Zarling. 1993. Nature Genet. 3: 365-372.
checking ... Filament formation is monitored by band shift assay in a 0.8% agarose gel. In this assay, adenosine 5'-triphosphate
Nucleoprotein filaments formed in the presence of (ATPγS) produce a slower band on an agarose gel that is more compact than uncoated ssDNA.

The formation of recombinant hybrids is monitored by standard methods (Golub et al. 1992
Nucl. Acids. Res. 20: 3121-3125; Golub et al. 1993. PNAS USA 90: 7186-71.
91; Belotserkovskii et al. 1998; Hsieh et al. 1992. PNAS USA 89: 6492-6.
496; Sena & Zarling, 1993). For probe-target hybridization, the nuclear filament is mixed with the target DNA and then incubated at 37 ° C. for 1 hour. SD
The protein of the mixture is removed by the addition of S and the hybrid (probe-target complex) is separated from the free probe and plasmid by electrophoresis on an agarose gel. The product is visualized by chemiluminescence or autoradiography. Resistance to restriction enzyme digestion and transcription is an indicator of hybrid formation (Hsieh et al. 1992) and is used for both qualitative and quantitative assessment of hybridization.

The hybrid conjugate was extracted from the gel and treated with ampicillin-sensitive RecA.
It is introduced into a positive E. coli chain (for example, BB4) by electroporation. Recombinants with increased β-lactamase activity show an increase in active β-lactamase when first placed in plating with ampicillin 50 mg / ml to 500 mg / ml and selected. Plasmids in individual colonies that show increased resistance are purified and subjected to subsequent mutagenesis and expansion. The plasmid in each round of selection was isolated from the mutated (developed) cells,
Sequences are determined by standard methods and characterized for the location and number of mutations.

Example 2 Development of Domain-Specific Genes for Botulinum Neurotoxin Antibodies Single-chain Fv (scFv) consists of immunoglobulin _VL and _VH domains linked by a peptide spacer, thus minimizing antigen binding of the antibody Including domain. A fusion protein of the scFv and the phage plll minor coat protein allows expression of the scFv on the surface of the phage to which the antigen can bind.
Five copies of plll are expressed on the surface of the phage, and therefore up to five scFvs can be expressed per phage particle (Barbas et al. PNAS USA 89: 4457-4461).
; Zebedee et al. 1992. PNAS USA 89: 3175-3179; Burton et al. 1991. PNAS US
A 88: 10134-10137).

To generate new antibodies from the phage antibody system, mice are immunized with a heat-inactive botulinum toxin. Spleen cells collected from these mice are used as a source of immunoglobulin mDNA that can be reverse transcribed into immunoglobulin cDNA.
The antibody _VL and _VH domains of the cDNA are isolated by PCR and spliced together with the appropriate oligonucleotide linkers into a scFv construct to create a scFv fusion protein library linked to a plll frame (Pharmacia, Piscataway,
NJ). The scFv phage library is screened to select phage that bind to the inactive botulinum neurotoxin.

To develop the affinity of the scFV, probes are synthesized and targeted to light and heavy chain CDRs. Each probe has a sequence that is degenerate for the corresponding CDR, but homologous to the framework region for the homology clamp (FIG. 8). The probe is conjugated to RecA and forms a nucleoprotein filament as described in Example 1. The filament is hybridized to the purified scFv phagemid DNA to form a hybrid complex. The complex is transformed into a recombinant E. coli strain (eg, BB4) to allow strand exchange. Bacteria are transformed with helper phage to collect and encompass the scFv phagemid containing the mutagenized or expanded CDR regions.

After each round of development, phage with scFv are screened for increased affinity binding with botulinum neurotoxin. Selection of phage with high affinity scFV is performed by continuously increasing the ionic strength and / or temperature of the bound state and measuring the dissociation constant. This procedure is repeated to develop an antibody phage library. After repeated mutagenesis, the phage that binds the botulinum toxin most strongly are selected.

Example 3 Development of Domain-Specific Genes of Antibodies to Bacillus Spores Vaccination against the antigenic component of Bacillus anthracis, the etiological agent of B. anthracis, is poorly effective and does not provide long-term immunity. The virulent form of Bacillus anthracis comprises a capsule antigenic form of α-polypeptide-D-glutamic acid (the most natural amino acid is L-type). The presence of the D-glutamate polypeptide in this capsule renders the macrophage phagocytic activity of Bacillus anthracis ineffective, thereby abolishing its effective function for the immune response. Gram-positive bacteria (eg, Bacillus anthracis) also contain teichoic acid as a cell wall composition. To develop a composition for the prevention and treatment of anthrax, an antibody phage library is screened for reactivity of D-glutamic acid to the α-polypeptide and teichoic acid. The sequence of the phage carrying these scFv clones is determined to define the CDR and frame regions. Probes were synthesized based on the inducible sequence and the light and heavy chains sc for tecoyl or D-glutamic acid
Targets the CDR regions of the Fv. The CDR region target hybrid is
Transformation with helper phage in E. coli with high recombination frequency allows for entrapment and expression. After the screen to remove unexpanded scFv phage, the procedure for library evolution is repeated. In the manner described above (Mayforth, 1993), the developed scFv is used for the construction of bispecific antibodies. One arm of the antigen-binding site of the bispecific antibody is taylcoic acid-specific, and the other arm is an alpha polypeptide.
-Specific for D-glutamic acid.

[Brief description of the drawings]

FIG. 1 shows domain-specific gene development (DSGE) by recombinase-mediated complementary single-stranded DNA targeting (cssDNA or targeting polynucleotide).

FIG. 2 shows a natural gene targeting assay (double D loop formation).

FIG. 3A shows probe target hybrid upregulated homologous recombination in bacteria. Panel A: Schematic for the EHR assay used in Panel B.

FIG. 3B shows probe target hybrid upregulated homologous recombination in bacteria. Panel B: cssDNA probe in E. coli: data for enhanced homologous recombination (EHR) of target hybrids.

FIG. 4 shows the design of a DSGE DNA probe.

FIG. 5 shows a targeting polynucleotide for expanding the CDR region of scFv into botulinum neurotoxin.

FIG. 6 shows a flow chart of steps in developing a single-chain scFv antibody phage library by DSGE.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Gurucharan Lady United States 94063 Redwood, California・ City, Marshall Street 1405, Apartment Number 510 (72) Inventor Susma Patty 94024 Los Angeles, CA, United States Amber Lane 816 F-Term (Reference) 4B024 AA01 AA20 BA80 CA01 CA04 CA07 CA09 CA11 DA02 DA05 DA11 EA03 EA04 GA11 GA25 HA01 4B064 AG01 CA01 CA19 CC24 DA01

Claims (24)

[Claims] 1. A method for developing a domain-specific gene of a target nucleic acid sequence encoding a desired amino acid sequence, comprising providing a plurality of pairs of single-stranded targeting polynucleotides, the pairs being substantially complementary to each other. And each comprising a homologous clamp that substantially corresponds to or is substantially complementary to a predetermined sequence of the target nucleic acid sequence encoding a domain of the protein; The pair comprises a library of mismatches between the targeting polynucleotide and the sequence and a recombinase,
A method comprising forming a library of modified target nucleic acid sequences. 2. The method of claim 1, further comprising providing a second plurality of pairs of single-stranded targeting polynucleotides, either simultaneously or subsequently, wherein said pairs are substantially complementary to each other. And each substantially corresponds to or is substantially complementary to a predetermined second sequence of the target nucleic acid sequence encoding a second domain of the protein. A homologous clamp, wherein the second plurality of pairs comprises a library of mismatches between the targeting polynucleotide and the second sequence and a recombinase to form a library of modified target nucleic acid sequences. 2. The method of claim 1, comprising: 3. A method for developing a domain-specific gene, comprising: a) a target nucleic acid encoding a desired amino acid sequence and a pair of single-stranded targeting polynucleotides, wherein the pair is substantially complementary to each other. And each substantially corresponds to a predetermined sequence of said target nucleic acid sequence encoding a domain of said protein,
(Including a homologous clamp that is substantially complementary) and a recombinase to form a recombinant intermediate; b) contacting the intermediate with a single-stranded exonuclease or a ligation-specific nuclease to cleave Or forming an open-ended target nucleic acid; c) re-assembling and recombining the truncated or open-ended target nucleic acid to create a library of modified target nucleic acid sequences. 4. In addition, d) the target nucleic acid encoding the desired amino acid sequence and a second pair of single-stranded targeting polynucleotides, wherein the pair is substantially complementary to each other. , Which are not substantially complementary to the first polynucleotide, and each substantially correspond to or are substantially complementary to a predetermined second sequence of the nucleic acid encoding a second domain of the protein. A homologous clamp) and a recombinase to form a recombinant intermediate; e) contacting the intermediate with a single-stranded exonuclease or a ligation-specific nuclease to generate a cut or open-ended 4. The method of claim 3, comprising forming a target nucleic acid; f) re-assembling and recombining the truncated or open-ended target nucleic acid to create a library of modified target nucleic acid sequences. 5. A method for creating a pool of variant nucleic acid sequences of a preselected target nucleic acid sequence in an external chromosomal sequence, comprising: a) the external chromosomal sequence comprising at least one recombinase and one of a plurality of pairs.
A single-stranded targeting polynucleotide, wherein the pair is substantially complementary to each other and each comprises a homologous clamp that substantially corresponds to or is substantially complementary to a preselected target nucleic acid sequence Wherein the plurality of pairs comprises a library of mismatches between the targeting polynucleotide and the target nucleic acid sequence to form a library of modified external chromosomal sequences; b) the modified Repeating step a) for said library of external chromosomal sequences;
A method that includes: 6. The method of claim 6, further comprising: d) adding at least one recombinase and a second plurality of single-stranded targeting polynucleotides to the external chromosomal sequence, wherein the pairs are substantially complementary to each other. A homologous clamp that is not substantially complementary to the first plurality of polynucleotides, and each substantially corresponds to or is substantially complementary to a second preselected target nucleic acid sequence. Wherein said second plurality of pairs comprises a library of mismatches between said targeting polynucleotide and said second target nucleic acid sequence, simultaneously or continuously, and wherein said modified external chromosome Forming a library of sequences; e) repeating step d) for said library of modified external chromosomal sequences;
6. The method of claim 5, comprising: 7. A method for creating a pool of variant nucleic acid sequences of a preselected target nucleic acid sequence in a chromosomal sequence, comprising: a) providing said chromosomal sequence with at least one recombinase and a plurality of pairs of single-stranded targets. A polynucleotide (the pair is substantially complementary to each other and each comprises a homologous clamp that substantially corresponds to or is substantially complementary to a preselected target nucleic acid sequence; A plurality of pairs, including a library of mismatches between the targeting polynucleotide and the target nucleic acid sequence) to form a library of modified chromosomal sequences; Repeating step a) for the library;
A method that includes: 8. The method of claim 1, further comprising: d) adding to said chromosomal sequence at least one recombinase and a second plurality of single-stranded targeting polynucleotides, wherein said pairs are substantially complementary to each other;
A homologous clamp that is not substantially complementary to the first plurality of polynucleotides, and each substantially corresponds to or is substantially complementary to a second preselected target nucleic acid sequence. Wherein the second plurality of pairs comprises a library of mismatches between the targeting polynucleotide and the second target nucleic acid sequence, simultaneously or continuously, and wherein the modified chromosomal sequence 8. The method of claim 7, comprising forming a library; e) repeating step d) for said library of modified chromosomal sequences. 9. The method of claim 1, 2, 3, or 4, further comprising repeating the method on the library of modified target nucleic acid sequences. 10. The method of claim 1, further comprising introducing said library of modified target nucleic acid sequences into a cell to form a cell library comprising a variant nucleic acid sequence. , 6, 7 or 8. 11. The method of claim 10, further comprising expressing said library of modified target nucleic acid sequences to create a pool of variant amino acid sequences. 12. The method of claim 10 or 11, further comprising selecting cells that contain the modified target nucleic acid sequence having the desired activity. 13. The method of claim 10 or 11, further comprising selecting cells containing the modified target nucleic acid sequence having a desired phenotype. 14. The method of claim 11, further comprising selecting said pool of variant amino acid sequences. 15. The method of claim 10, wherein said recombinase is removed prior to said introduction.
The method of 11, 12 or 13. 16. The method according to claim 1, wherein said cells are eukaryotic cells.
, 7, 8, 9, 10, 11, 12, 13, 14, or 15. 17. The method of claim 1,2,3,4,5,6, wherein said cells are prokaryotic cells.
, 7, 8, 9, 10, 11, 12, 13, 14, or 15. 18. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 wherein the targeting polynucleotide is coated with the recombinase.
, 14, 15, 16 or 17. 19. The recombinase of claim 1,2,3,4,5,6,7,8,9,10,11,12,13,1 wherein the recombinase is a prokaryotic recombinase.
The method of 4, 15, 16, 17 or 18. 20. The recombinase of claim 1,2,3,4,5,6,7,8,9,10,11,12,13,1 wherein the recombinase is one eukaryotic cell recombinase.
The method of 4, 15, 16, 17 or 18. 21. The variant amino acid sequence comprises a plurality of amino acid substitutions.
Method 1. 22. The method of claim 1,2,3,4,5,6,7,8,9,10,11,12,13 wherein at least one said complementary single-stranded nucleic acid further comprises a chemical substituent.
, 14, 15, 16, 17, 18, 19, 20 or 21. 23. The method of claim 1, wherein the target amino acid sequence comprises a complementarity determining region.
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
The method of 17, 18, 19, 20, 21, or 22. 24. The method according to claim 1, wherein said target nucleic acid sequence comprises an expression vector.
, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17
, 18, 19, 20, 21, 22, or 23.