CN108893450B - BHK21 cell population reconstructed by innate immune system and application of cell clone in virus amplification - Google Patents

BHK21 cell population reconstructed by innate immune system and application of cell clone in virus amplification Download PDF

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CN108893450B
CN108893450B CN201810613327.4A CN201810613327A CN108893450B CN 108893450 B CN108893450 B CN 108893450B CN 201810613327 A CN201810613327 A CN 201810613327A CN 108893450 B CN108893450 B CN 108893450B
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golden hamster
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CN108893450A (en
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冯磊
陈丽
侯继波
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Jiangsu Academy of Agricultural Sciences
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Abstract

The invention discloses a method for enhancing virus proliferation, and a BHK21 cell population reconstructed by an innate immune system is constructed and used for proliferating viruses. The method disclosed by the invention opens a new thought for virus propagation, expands the application range of the BHK21 cells, and lays a solid foundation for the development of vaccines capable of preventing and treating viruses.

Description

BHK21 cell population reconstructed by innate immune system and application of cell clone in virus amplification
Technical Field
The invention belongs to the technical field of biotechnology and biological product engineering for veterinary use, and particularly relates to a BHK21 cell population for randomly knocking out effector protein molecules of an innate immune system and application of a cell clone proliferation virus of the BHK21 cell population.
Background
The BHK21 cells are the common host cells for livestock and poultry vaccine virus proliferation at present, and various large research institutions and veterinary vaccine production enterprises are all conducting researches on seed cell screening, cell suspension culture, virus proliferation processes and the like for the cell proliferation of various viruses, and the method is a current research hotspot. The innate immune system of the host cell is a body defense mechanism that is generated at an early stage of phylogeny and occurs at the initial stage of the host's anti-infection response to perform immune functions, and functions to resist infection, remove harmful components from the body, autoimmunity, and transplant rejection. Host cells sense pathogen invasion through their pathogen-associated pattern recognition receptors, which in turn initiate interferons and a range of cytokines to counteract pathogen invasion. These model molecules are expressed in different types of cells or in different regions of organelles, such as cell membranes, endosomal membranes, lysosomal membranes, cytoplasm, etc., and are primarily responsible for monitoring various component molecules of pathogens to initiate inflammatory responses and antiviral immunity. Specific model molecules include Toll-like receptors, RIG-I-like receptors, NOD-like receptors, DAI molecules, AIM2 molecules, and the like. For the large-scale proliferation of the virus, the signal transduction pathway of the innate immune system is reduced, and the inhibition of the antagonistic action of the virus proliferation is beneficial to the efficient amplification of the virus. By using a genome editing technology to randomly combine and knock out signal molecules in a BHK21 cell innate immune system, the innate immune response caused by exogenous virus infection is reduced, and a good intracellular biological environment is provided for high-titer propagation of various viruses in BHK21 cells.
Viral infection is effectively inhibited by targeting viral genomic DNA often using the CRISPR/Cas9 system in the prior art. According to the technical principle of CRISPR/Cas9, a gRNA of a PRV-conservative UL30 gene is designed, and a PK-15 cell line capable of stably expressing sgRNA and Cas9 endonuclease is successfully constructed. The results show that after the cell is infected by PRV, the editing effect of the CRISPR/Cas9 system on the PRV UL30 gene can cause the base deletion or insertion of the UL30 gene, and the proliferation of the PRV in the cell can be obviously inhibited.
ERp57 is a member of the protein disulphide isomerase family within the intracellular endoplasmic reticulum, and can catalyse the synthesis of protein disulphide bonds to facilitate protein folding. In order to research the influence of erp57 gene knockout on influenza virus replication, researchers construct guide RNA (gRNA) knockout plasmid pGEM-erp57gRNA of erp57 gene by using CRISPR/Cas9 technology, co-transfect the plasmid and pCas9-EGFP into human lung adenocarcinoma cell A549, screen single cell clone strains by using a flow sorting and limiting dilution method, and screen and obtain an A549 cell line of erp57 gene knockout by using target genome sequencing and western blot detection. The erp57 gene knockout cell line has no significant difference from the growth kinetics of wild-type cells. In addition, H1N1 subtype influenza viruses were infected with wild-type and erp57 gene knockout cell lines, respectively, to determine the effect of erp57 gene knockout on the level of replication of influenza viruses. The results show that influenza replication in erp57 gene knockout cells is significantly inhibited.
In view of the prior art, the inventors have found that the gene knockout method is commonly used by those skilled in the art to inhibit the proliferation of viruses, and have not found the application of the BHK21 cell population and its cell clone reconstructed from the innate immune system in the aspect of virus proliferation.
Disclosure of Invention
The present invention provides a method for enhancing virus proliferation, which is characterized in that the virus is proliferated in BHK21 cells in which innate immune system reconstitution has been performed.
The present invention provides a method for enhancing virus proliferation, wherein a signaling pathway in the innate immune system in BHK21 cells is reduced.
The present invention provides a method for enhancing virus proliferation, wherein the reduction of the signaling pathway is achieved by gene knockout of a plurality of signaling molecules in the innate immune system of BHK21 cells.
The present invention provides a method for enhancing virus proliferation, wherein the reduction of the signaling pathway is achieved by randomly combinatorial gene knockout of a plurality of signaling molecules in the innate immune system of BHK21 cells.
The present invention also provides a method for enhancing viral proliferation, wherein the plurality of signal molecules are selected from one or more of the group consisting of recognition receptor proteins, effector proteins, stress proteins, protein kinases, interferons, and their downstream regulatory products.
The invention provides a method for enhancing virus proliferation, which is further characterized in that gene knockout of various signal molecules is realized through a CRISPR/Cas9 system.
The invention provides a method for enhancing virus proliferation, which is further characterized in that the gene knockout of a plurality of signal molecules comprises the steps of selecting at least two target sequences of the plurality of signal molecules and designing a pair of DNA oligo primers aiming at each target sequence.
The present invention also provides a method for enhancing virus propagation, wherein the target sequence is obtained by analyzing a genomic sequence of hamster with golden color.
The present invention provides a method for enhancing virus propagation, which is further characterized in that a pUC-gRNA-library is constructed using the DNA oligo primer.
The present invention provides a method for enhancing viral propagation, further characterized in that the DNA oligo primer is selected from the group consisting of sequences 1 to 446.
The present invention provides a method for enhancing virus proliferation, wherein the virus is selected from the group consisting of pseudorabies virus, japanese encephalitis virus, foot and mouth disease virus, rabies virus, and newcastle disease virus.
The present invention provides a method for enhancing virus proliferation, which is further characterized by establishing a BHK21 cell population in which the innate immune system is reconstituted.
The invention provides a method for enhancing virus proliferation, which is further characterized by screening a BHK21 cell population reconstructed by an innate immune system to obtain the best BHK21 cell reconstructed by the innate immune system for virus proliferation.
The invention also provides a method for enhancing virus proliferation, which is characterized in that the obtained progeny virus proliferated from the BHK21 cell reconstructed by the optimal innate immune system is diluted to 10-8Positive lesions still developed.
The invention provides a method for enhancing virus proliferation, which is further characterized in that a gRNA expression vector framework is adopted to construct a pUC-gRNA-library, and the gRNA expression vector framework contains a U6 promoter, an RNA support and a polyA signal sequence.
The present invention provides a method for enhancing virus propagation, which is further characterized in that a medium different from that used in the initial culture of BHK21 cells is used during transfection of BHK21 cells with a pUC-gRNA-library.
The present invention provides a method for enhancing virus propagation, which is further characterized in that Opti-MEM medium is used during transfection of BHK21 cells with the pUC-gRNA-library.
The invention provides a method for enhancing virus proliferation, which is further characterized in that a pUC-gRNA-library is used for transfecting BHK21 cells, and a dropwise adding mode is adopted to uniformly mix a transfection compound.
The invention provides a method of enhancing viral proliferation, further characterized in that the transfection complex comprises a polyethyleneimine reagent.
The invention provides a method for enhancing virus proliferation, which is further characterized in that a culture medium of an initial BHK21 cell is replaced before transfection, and then transfection is carried out after a period of pre-culture.
The present invention provides a method for enhancing virus propagation, further characterized in that the cell clones obtained are subjected to at least one freeze-thaw cycle when the titer of a virus propagation sample is determined.
The present invention provides a method for enhancing virus proliferation, comprising:
firstly, analyzing genome sequences of golden hamster, aiming at respective gene sequences of recognition receptor protein, effector protein, stress protein, protein kinase, interferon and downstream regulation product thereof of innate immune system, selecting target sequences according to sequence recognition requirement of gene editing in CRISPR/Cas9 system; selecting at least two target sequences from each gene sequence; designing a pair of DNA oligo primers for each target sequence;
annealing each pair of target sequence primers to generate a DNA double strand with a sticky end; treating a gRNA expression vector skeleton by using restriction endonuclease to generate a cohesive end which is the same as a primer annealing product, and performing T4DNA ligase action to obtain a pUC-gRNA-library for a subsequent transfection experiment;
initial cell density of 1X 10 for seeding6BHK21 cells/ml were cultured overnight in 6-well plates and incubated for 10 min with the pre-transfection medium replaced with Opti-MEM medium; adding 100ul of Polyethyleneimine (PEI) reagent into 1ml of Opti-MEM, uniformly mixing, adding 1-10 ug of pCas9-IRES-GFP and pUC-gRNA-library plasmid vector into 1ml of Opti-MEM, uniformly mixing, and incubating for 5 minutes at room temperature; after incubation is finished, uniformly mixing the two mixed solutions in a dropwise adding mode, and incubating for 15 minutes at room temperature to form a PEI-DNA transfection complex; adding 2ml of the transfection complex into BHK21 cells in a 6-well plate in a dropwise adding mode for transfection; 8 hours after transfection, the transfection complex is removed, and the cell is continuously cultured for 24 hours by replacing the cell with a normal culture medium of BHK21 cells;
BHK21 cells 24 hours after transfection are used for cell sorting of a flow cytometer after digestion, dispersion, centrifugal recovery and PBS washing; before sorting, the cells are filtered by a sterile 600-mesh screen and then sorted by an up-flow cytometer, and the cell population with green fluorescence is sorted into a pore plate; after the cell growth is recovered, establishing a library and storing the library as a BHK21-KO-IM cell population;
sorting the BHK21-KO-IM cell population into a 96-well plate by a flow cytometer for culture, wherein each well is provided with one cell clone; after the growth is recovered, adjusting the cell density of each hole to be approximately the same, and manufacturing a compound plate for comparing the virus proliferation efficiency after the cells are amplified;
removing culture supernatant, and respectively inoculating 200ul of virus proliferation maintenance liquid containing the same; observing cytopathic effect every day, completing virus proliferation in 72 hours, and measuring the titer of each virus proliferation sample after 3 times of freeze thawing; if the cell pathological changes exist, the cell pathological changes are judged to be positive, otherwise, the cell pathological changes are negative, the virus multiplication capacities of different cell clones are compared, and the cell clones with the improved multiplication capacity are obtained.
A BHK21 cell population for innate immune system reconstitution constructed by the methods described above.
Use of a BHK21 cell population reconstituted from the innate immune system constructed by the method described above for propagating a virus.
The invention achieves the following beneficial effects:
(1) the invention establishes the BHK21 cell population reconstructed by the innate immune system, screens the cell population to obtain the BHK21 cell reconstructed by the optimal innate immune system, and proliferates a plurality of viruses.
(2) The invention adopts the randomly combined DNA oligo primer to construct a pUC-gRNA-library and construct a BHK21 cell population reconstructed by an innate immune system, thereby opening a new thought for virus proliferation.
(3) The BHK21 cell can be used for propagating more viruses and providing a propagation mode for the viruses.
(4) The establishment of a pUC-gRNA-library provides a new idea for constructing BHK21 cells reconstructed by an innate immune system.
(5) The invention proliferates the virus by the BHK21 cell clone reconstructed by the innate immune system, breaks through the method that the virus is proliferated only by the seed cell screening, cell suspension culture and virus proliferation processes in the prior art, and provides a new method for virus proliferation.
(6) The CRISPR/Cas9 system is adopted for gene knockout, gene editing can be efficiently completed, and a target vector for transfecting cells is obtained.
(7) When the BHK21 cell population reconstructed by the innate immune system obtained by the invention is used for propagating viruses, the propagation titer of the viruses can be greatly improved, the large-scale propagation of the viruses is facilitated, and vaccines are obtained for immunization.
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Other features and advantages of the present invention will become apparent from the following detailed description when taken in conjunction with the accompanying drawings, in which:
FIG. 1 shows a schematic diagram comparing the efficiency of propagating pseudorabies virus (PRV) between BHK21 mother cells and 6 cell clones selected according to the present invention.
FIG. 2 is a schematic diagram showing the comparison of the efficiency of propagating Japanese Encephalitis Virus (JEV) between BHK21 mother cells and 9 cell clones selected according to the present invention.
FIG. 3 shows a schematic diagram comparing the efficiency of propagating Newcastle Disease Virus (NDV) between BHK21 mother cells and 9 cell clones selected according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example 1 acquisition of BHK21-KO-IM cell population and Generation of Single cell clones
1. Construction of a pUC-gRNA library
The genome sequence of golden hamster is analyzed, and the target sequence is selected according to the sequence recognition requirement of gene editing in the CRISPR/Cas9 system aiming at the respective gene sequences of recognition receptor protein, effector protein, stress protein, protein kinase, interferon, downstream regulation product thereof and the like of the innate immune system. Typically, at least two target sequences are selected for each gene sequence. For each target sequence, a pair of DNA oligo primers is designed. The primers used were primers of SEQ ID Nos. 1 to 446.
Each pair of target sequence primers is annealed separately to generate a DNA double strand having a cohesive end. The gRNA expression vector backbone (containing the U6 promoter, RNA scaffold, polyA signal sequence) was treated with the restriction enzyme BbsI (available from NEB biotechnology) to generate cohesive ends identical to the primer annealing products. A pUC-gRNA-library was obtained by T4DNA ligase (purchased from TAKARA Biotech) and used for subsequent transfection experiments.
2. Transfection of BHK21 cells
Initial cell density of 1X 10 for seeding6BHK21 cells were cultured overnight in 6-well plates per ml. The pre-transfection medium was replaced with Opti-MEM medium (purchased from Invitrogen) and incubated for 10 minutes. 100ul PEI reagent (purchased from Sigma) was added to 1ml of Opti-MEM and mixed, 1-10 ug of pCas9-IRES-GFP and pUC-gRNA-library plasmid vector were added to 1ml of Opti-MEM and mixed, and incubated at room temperature for 5 minutes. And after the incubation is finished, uniformly mixing the two mixed solutions in a dropwise adding mode, and incubating for 15 minutes at room temperature to form the PEI-DNA transfection complex. The transfection was carried out by adding 2ml of the above transfection complex dropwise to BHK21 cells in a 6-well plate. 8 hours after transfection, the transfection complex was removed and the medium was replaced with normal medium for BHK21 cells and cultured for another 24 hours.
3. Screening of BHK-KO-IM cell population
BHK21 cells 24 hours after transfection were used for flow cytometry cell sorting after digestion, dispersion, recovery by centrifugation, and washing with PBS. Before sorting, the cells are filtered by a sterile 600-mesh screen and then sorted by an up-flow cytometer, and the cell population with green fluorescence is sorted into a pore plate. When the cell growth is recovered, the cell is stored as BHK21-KO-IM cell group by establishing a library.
4. Obtaining of clone of BHK21-KO-IM cell
The BHK21-KO-IM cell population was sorted into 96-well plates for culture by flow cytometry, one cell clone per well. And after the cell clone grows and recovers, performing a double plate operation on the cell clone for cell preservation and virus proliferation comparison experiments.
Example 2 screening of PRV high-yielding BHK21 cell clones and application thereof to viral propagation
1. The BHK21-KO-IM cell group is sorted by a flow cytometer to form single cell clones in a 96-well plate, 177 actual cell clones are obtained after the growth is recovered, the cell density of each well is adjusted to be approximately the same, and a compound plate is manufactured after the cells are amplified for comparing the virus propagation efficiency.
2. The culture supernatant was removed, and 200 ul/well of a virus growth maintenance solution containing the same amount of porcine pseudorabies virus (PRV) was inoculated. The cytopathic effect is observed every day, the virus proliferation is completed in 72 hours, and the titer of virus proliferation samples in each hole is determined after 3 times of freeze thawing. Specifically, 10 was done for each virus sample-6、10-7、10-8After dilution, the cell is inoculated on a common monolayer BHK21 cell, and the cytopathic condition is observed after 72 hours, if the cell has typical cytopathic condition, the cell is judged to be positive, otherwise, the cell is negative. The results of comparing the virus proliferation capacities of different cell clones are shown in tables 1 and 2. As a result, PRV progeny viruses proliferated from cell clones 1-C9, 1-E2, 1-E11, 1-G2, 2-B10 and 2-D8 were diluted to 10-8Typical positive lesions can still be generated, indicating that several cell clones have higher virus proliferation capacity than other cell clones.
3. The 6 cell clones selected above were amplified, and suspension growth acclimatization, high-density cell culture and PRV suspension proliferation comparison were performed, and the results are shown in FIG. 1, in which the virus proliferation potency of the 6 cell clones was significantly higher than that of BHK21 mother cells, and the virus proliferation potency of cell clones 1-E2, 1-G2 and 2-D8 all exceeded 9.0lgTCID50The virus propagation titer of BHK21 mother cells was only 7.45 lgTCID/ml50/ml。
Example 3 screening of high-yield BHK21 cell clone of JEV and application of cell clone in virus propagation
1. The BHK21-KO-IM cell group is sorted by a flow cytometer to form single cell clones in a 96-well plate, 177 actual cell clones are obtained after the growth is recovered, the cell density of each well is adjusted to be approximately the same, and a compound plate is manufactured after the cells are amplified for comparing the virus propagation efficiency.
2. The culture supernatant was removed, and 200ul of a virus growth maintenance solution containing the same amount of Japanese Encephalitis Virus (JEV) was inoculated thereto. The cytopathic effect is observed every day, the virus proliferation is completed within 96 hours, and the titer of each virus proliferation sample is determined after 3 times of freeze thawing. The virus titer detection method is used for carrying out 10 steps on a virus proliferation sample according to the quality standard requirement of animal biological products of Ministry of agriculture-4、10-5、10-6、10-7、10-8Diluting, inoculating to common single layer BHK21 cell, adsorbing for 90 min, adding 1% methylcellulose covering material, and continuingCulture, stain count after 5 days, calculate plaque formation units, expressed as lgPFU/ml.
3. The results of the suspension growth acclimatization, high-density cell culture and JEV suspension proliferation comparison of the 9 selected cell clones are shown in FIG. 2, the virus proliferation capacity of the 9 cell clones is significantly higher than that of the BHK21 mother cells, wherein the virus proliferation titer of the cell clones 1-G9 and 2-D2 is over 6.5lgPFU/ml, while the virus proliferation titer of the BHK21 mother cells is only 4.75 lgPFU/ml.
Example 4 screening of high-yield BHK21 cell clone of NDV and its virus propagation application
1. The BHK21-KO-IM cell group is sorted by a flow cytometer to form single cell clones in a 96-well plate, 177 actual cell clones are obtained after the growth is recovered, the cell density of each well is adjusted to be approximately the same, and a compound plate is manufactured after the cells are amplified for comparing the virus propagation efficiency.
2. The culture supernatant was removed, and 200 ul/well of a virus growth maintenance solution containing the same amount of Newcastle Disease Virus (NDV) was inoculated, respectively. The cytopathic effect is observed every day, the virus proliferation is completed in 72 hours, and the titer of virus proliferation samples in each hole is determined after 3 times of freeze thawing. The virus titer detection method is used for carrying out 10 steps on a virus proliferation sample according to the quality standard requirement of animal biological products of Ministry of agriculture-5、10-6、10-7、10-8Diluting, inoculating to monolayer chicken embryo fibroblast, culturing, calculating virus titer according to pathological changes after 3 days, and collecting lgTCID50Expressed in ml.
3. The results of suspension growth acclimatization, high-density cell culture and NDV suspension proliferation comparison of the 9 selected cell clones are shown in figure 3, the virus proliferation capacity of the 9 cell clones is remarkably higher than that of BHK21 mother cells, wherein the virus proliferation titers of the cell clones 1-C2, 1-H3, 2-D9 and 2-G8 are all over 9.0lgTCID50The virus propagation titer of BHK21 mother cells was only 6.75 lgTCID/ml50/ml。
In the present invention, the BHK21 — KO-IM cells are used to clone and propagate pseudorabies virus (PRV), Japanese Encephalitis Virus (JEV), and Newcastle Disease Virus (NDV) viruses, but the present invention is not limited to PRV, JEV, and NDV viruses, and the PRV, JEV, and NDV viruses are merely exemplified, and the virus propagation applications of the present disclosure also include Foot and Mouth Disease Virus (FMDV), Rabies Virus (RV), and the like.
It should be emphasized that the above-described embodiments of the present invention are merely possible examples of implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiments of the disclosure without departing from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.
Figure BDA0001696057300000111
Sequence listing
<110> agricultural science and academy of Jiangsu province
<120> BHK21 cell population reconstructed by innate immune system and application of cell clone in virus amplification
<130> DC18FM842
<141> 2018-06-14
<160> 446
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Golden hamster
<400> 1
caccgagggt aggtccttgg gcaca 25
<210> 2
<211> 25
<212> DNA
<213> Golden hamster
<400> 2
aaactgtgcc caaggaccta ccctc 25
<210> 3
<211> 25
<212> DNA
<213> Golden hamster
<400> 3
caccgccagt tggtttggca tacac 25
<210> 4
<211> 25
<212> DNA
<213> Golden hamster
<400> 4
aaacgtgtat gccaaaccaa ctggc 25
<210> 5
<211> 25
<212> DNA
<213> Golden hamster
<400> 5
caccgcaggt caccatggcc aatgg 25
<210> 6
<211> 25
<212> DNA
<213> Golden hamster
<400> 6
aaacccattg gccatggtga cctgc 25
<210> 7
<211> 25
<212> DNA
<213> Golden hamster
<400> 7
caccgacacc aagacgtacc tggaa 25
<210> 8
<211> 25
<212> DNA
<213> Golden hamster
<400> 8
aaacttccag gtacgtcttg gtgtc 25
<210> 9
<211> 25
<212> DNA
<213> Golden hamster
<400> 9
caccggaggt gtgggagcca agaga 25
<210> 10
<211> 25
<212> DNA
<213> Golden hamster
<400> 10
aaactctctt ggctcccaca cctcc 25
<210> 11
<211> 25
<212> DNA
<213> Golden hamster
<400> 11
caccgccaat gtacggtgag acatg 25
<210> 12
<211> 25
<212> DNA
<213> Golden hamster
<400> 12
aaaccatgtc tcaccgtaca ttggc 25
<210> 13
<211> 25
<212> DNA
<213> Golden hamster
<400> 13
caccgccccg ggataaactc tggat 25
<210> 14
<211> 25
<212> DNA
<213> Golden hamster
<400> 14
aaacatccag agtttatccc ggggc 25
<210> 15
<211> 25
<212> DNA
<213> Golden hamster
<400> 15
caccgcattg ccgccaacat catcc 25
<210> 16
<211> 25
<212> DNA
<213> Golden hamster
<400> 16
aaacggatga tgttggcggc aatgc 25
<210> 17
<211> 25
<212> DNA
<213> Golden hamster
<400> 17
caccgcgcca catcatttcc attcc 25
<210> 18
<211> 25
<212> DNA
<213> Golden hamster
<400> 18
aaacggaatg gaaatgatgt ggcgc 25
<210> 19
<211> 25
<212> DNA
<213> Golden hamster
<400> 19
caccgccaaa ccaacgtcac cctgc 25
<210> 20
<211> 25
<212> DNA
<213> Golden hamster
<400> 20
aaacgcaggg tgacgttggt ttggc 25
<210> 21
<211> 25
<212> DNA
<213> Golden hamster
<400> 21
caccggttca tcagaggact gcatc 25
<210> 22
<211> 25
<212> DNA
<213> Golden hamster
<400> 22
aaacgatgca gtcctctgat gaacc 25
<210> 23
<211> 25
<212> DNA
<213> Golden hamster
<400> 23
caccggtctg cagtctcttt aaagt 25
<210> 24
<211> 25
<212> DNA
<213> Golden hamster
<400> 24
aaacacttta aagagactgc agacc 25
<210> 25
<211> 25
<212> DNA
<213> Golden hamster
<400> 25
caccggcatc taggctgacg tcaca 25
<210> 26
<211> 25
<212> DNA
<213> Golden hamster
<400> 26
aaactgtgac gtcagcctag atgcc 25
<210> 27
<211> 25
<212> DNA
<213> Golden hamster
<400> 27
caccgatgtg gcagtctgca ttcaa 25
<210> 28
<211> 25
<212> DNA
<213> Golden hamster
<400> 28
aaacttgaat gcagactgcc acatc 25
<210> 29
<211> 25
<212> DNA
<213> Golden hamster
<400> 29
caccgaggtc agagtgttgt atctc 25
<210> 30
<211> 25
<212> DNA
<213> Golden hamster
<400> 30
aaacgagata caacactctg acctc 25
<210> 31
<211> 25
<212> DNA
<213> Golden hamster
<400> 31
caccgagagc acctgcaagt gtgga 25
<210> 32
<211> 25
<212> DNA
<213> Golden hamster
<400> 32
aaactccaca cttgcaggtg ctctc 25
<210> 33
<211> 25
<212> DNA
<213> Golden hamster
<400> 33
caccggcaca ctatttcagt atagc 25
<210> 34
<211> 25
<212> DNA
<213> Golden hamster
<400> 34
aaacgctata ctgaaatagt gtgcc 25
<210> 35
<211> 25
<212> DNA
<213> Golden hamster
<400> 35
caccgctgca tgaggttatc aatga 25
<210> 36
<211> 25
<212> DNA
<213> Golden hamster
<400> 36
aaactcattg ataacctcat gcagc 25
<210> 37
<211> 25
<212> DNA
<213> Golden hamster
<400> 37
caccggagct gaacctgagt tacaa 25
<210> 38
<211> 25
<212> DNA
<213> Golden hamster
<400> 38
aaacttgtaa ctcaggttca gctcc 25
<210> 39
<211> 25
<212> DNA
<213> Golden hamster
<400> 39
caccgagttc ttaggggtgc tcagt 25
<210> 40
<211> 25
<212> DNA
<213> Golden hamster
<400> 40
aaacactgag cacccctaag aactc 25
<210> 41
<211> 25
<212> DNA
<213> Golden hamster
<400> 41
caccgccagc acaaacagcg tcttg 25
<210> 42
<211> 25
<212> DNA
<213> Golden hamster
<400> 42
aaaccaagac gctgtttgtg ctggc 25
<210> 43
<211> 25
<212> DNA
<213> Golden hamster
<400> 43
caccgctaaa tgtctgagac ctacc 25
<210> 44
<211> 25
<212> DNA
<213> Golden hamster
<400> 44
aaacggtagg tctcagacat ttagc 25
<210> 45
<211> 25
<212> DNA
<213> Golden hamster
<400> 45
caccgcggaa ggcctgatga ttgga 25
<210> 46
<211> 25
<212> DNA
<213> Golden hamster
<400> 46
aaactccaat catcaggcct tccgc 25
<210> 47
<211> 25
<212> DNA
<213> Golden hamster
<400> 47
caccgaggca atggctccag taaga 25
<210> 48
<211> 25
<212> DNA
<213> Golden hamster
<400> 48
aaactcttac tggagccatt gcctc 25
<210> 49
<211> 25
<212> DNA
<213> Golden hamster
<400> 49
caccgtggtt caggcgctac caagc 25
<210> 50
<211> 25
<212> DNA
<213> Golden hamster
<400> 50
aaacgcttgg tagcgcctga accac 25
<210> 51
<211> 25
<212> DNA
<213> Golden hamster
<400> 51
caccggaagc actaggtcga tccgt 25
<210> 52
<211> 25
<212> DNA
<213> Golden hamster
<400> 52
aaacacggat cgacctagtg cttcc 25
<210> 53
<211> 25
<212> DNA
<213> Golden hamster
<400> 53
caccggttct tgaacgtacg gacgc 25
<210> 54
<211> 25
<212> DNA
<213> Golden hamster
<400> 54
aaacgcgtcc gtacgttcaa gaacc 25
<210> 55
<211> 25
<212> DNA
<213> Golden hamster
<400> 55
caccgagtcg cttctgttgg atgcc 25
<210> 56
<211> 25
<212> DNA
<213> Golden hamster
<400> 56
aaacggcatc caacagaagc gactc 25
<210> 57
<211> 25
<212> DNA
<213> Golden hamster
<400> 57
caccgcaggt accggaggaa cagtg 25
<210> 58
<211> 25
<212> DNA
<213> Golden hamster
<400> 58
aaaccactgt tcctccggta cctgc 25
<210> 59
<211> 25
<212> DNA
<213> Golden hamster
<400> 59
caccgattga agaaaatcct gcact 25
<210> 60
<211> 25
<212> DNA
<213> Golden hamster
<400> 60
aaacagtgca ggattttctt caatc 25
<210> 61
<211> 25
<212> DNA
<213> Golden hamster
<400> 61
caccggtgct ctaccaggag ctaac 25
<210> 62
<211> 25
<212> DNA
<213> Golden hamster
<400> 62
aaacgttagc tcctggtaga gcacc 25
<210> 63
<211> 25
<212> DNA
<213> Golden hamster
<400> 63
caccgcagca gtggttctgg agtta 25
<210> 64
<211> 25
<212> DNA
<213> Golden hamster
<400> 64
aaactaactc cagaaccact gctgc 25
<210> 65
<211> 25
<212> DNA
<213> Golden hamster
<400> 65
caccggacca tggcaaccct gagtg 25
<210> 66
<211> 25
<212> DNA
<213> Golden hamster
<400> 66
aaaccactca gggttgccat ggtcc 25
<210> 67
<211> 25
<212> DNA
<213> Golden hamster
<400> 67
caccggctgc cctctgttgc cacaa 25
<210> 68
<211> 25
<212> DNA
<213> Golden hamster
<400> 68
aaacttgtgg caacagaggg cagcc 25
<210> 69
<211> 25
<212> DNA
<213> Golden hamster
<400> 69
caccgaggaa gcccgtagtc gatcc 25
<210> 70
<211> 25
<212> DNA
<213> Golden hamster
<400> 70
aaacggatcg actacgggct tcctc 25
<210> 71
<211> 25
<212> DNA
<213> Golden hamster
<400> 71
caccgacctg ggacaacagc acacc 25
<210> 72
<211> 25
<212> DNA
<213> Golden hamster
<400> 72
aaacggtgtg ctgttgtccc aggtc 25
<210> 73
<211> 25
<212> DNA
<213> Golden hamster
<400> 73
caccgctaag agcaaggcca agagg 25
<210> 74
<211> 25
<212> DNA
<213> Golden hamster
<400> 74
aaaccctctt ggccttgctc ttagc 25
<210> 75
<211> 25
<212> DNA
<213> Golden hamster
<400> 75
caccgccctt gccatccacg tttgt 25
<210> 76
<211> 25
<212> DNA
<213> Golden hamster
<400> 76
aaacacaaac gtggatggca agggc 25
<210> 77
<211> 25
<212> DNA
<213> Golden hamster
<400> 77
caccgaggaa ctgggcgatc cacac 25
<210> 78
<211> 25
<212> DNA
<213> Golden hamster
<400> 78
aaacgtgtgg atcgcccagt tcctc 25
<210> 79
<211> 25
<212> DNA
<213> Golden hamster
<400> 79
caccgaggga gaggtctgta aatgg 25
<210> 80
<211> 25
<212> DNA
<213> Golden hamster
<400> 80
aaacccattt acagacctct ccctc 25
<210> 81
<211> 25
<212> DNA
<213> Golden hamster
<400> 81
caccggtctg gcttatcctt cccgg 25
<210> 82
<211> 25
<212> DNA
<213> Golden hamster
<400> 82
aaacccggga aggataagcc agacc 25
<210> 83
<211> 25
<212> DNA
<213> Golden hamster
<400> 83
caccgactcc ccacgctaca ctctg 25
<210> 84
<211> 25
<212> DNA
<213> Golden hamster
<400> 84
aaaccagagt gtagcgtggg gagtc 25
<210> 85
<211> 25
<212> DNA
<213> Golden hamster
<400> 85
caccggtgta caggattgtt ccaga 25
<210> 86
<211> 25
<212> DNA
<213> Golden hamster
<400> 86
aaactctgga acaatcctgt acacc 25
<210> 87
<211> 25
<212> DNA
<213> Golden hamster
<400> 87
caccgcaccg aagcacagag tcacc 25
<210> 88
<211> 25
<212> DNA
<213> Golden hamster
<400> 88
aaacggtgac tctgtgcttc ggtgc 25
<210> 89
<211> 25
<212> DNA
<213> Golden hamster
<400> 89
caccgtaccc aaagaaggcg tcaag 25
<210> 90
<211> 25
<212> DNA
<213> Golden hamster
<400> 90
aaaccttgac gccttctttg ggtac 25
<210> 91
<211> 25
<212> DNA
<213> Golden hamster
<400> 91
caccgcctgg tcagctgaca gcatc 25
<210> 92
<211> 25
<212> DNA
<213> Golden hamster
<400> 92
aaacgatgct gtcagctgac caggc 25
<210> 93
<211> 25
<212> DNA
<213> Golden hamster
<400> 93
caccgtatgc agccagatga gacca 25
<210> 94
<211> 25
<212> DNA
<213> Golden hamster
<400> 94
aaactggtct catctggctg catac 25
<210> 95
<211> 25
<212> DNA
<213> Golden hamster
<400> 95
caccgccttt gacagtggca gcgtt 25
<210> 96
<211> 25
<212> DNA
<213> Golden hamster
<400> 96
aaacaacgct gccactgtca aaggc 25
<210> 97
<211> 25
<212> DNA
<213> Golden hamster
<400> 97
caccgacaca gtcttccgtg tgccc 25
<210> 98
<211> 25
<212> DNA
<213> Golden hamster
<400> 98
aaacgggcac acggaagact gtgtc 25
<210> 99
<211> 25
<212> DNA
<213> Golden hamster
<400> 99
caccggacag gacttgtcag caggc 25
<210> 100
<211> 25
<212> DNA
<213> Golden hamster
<400> 100
aaacgcctgc tgacaagtcc tgtcc 25
<210> 101
<211> 25
<212> DNA
<213> Golden hamster
<400> 101
caccgagttt acagaattgt ccccg 25
<210> 102
<211> 25
<212> DNA
<213> Golden hamster
<400> 102
aaaccgggga caattctgta aactc 25
<210> 103
<211> 25
<212> DNA
<213> Golden hamster
<400> 103
caccggctat caggaaggcg actct 25
<210> 104
<211> 25
<212> DNA
<213> Golden hamster
<400> 104
aaacagagtc gccttcctga tagcc 25
<210> 105
<211> 25
<212> DNA
<213> Golden hamster
<400> 105
caccgtgtca tcccagcaca cccct 25
<210> 106
<211> 25
<212> DNA
<213> Golden hamster
<400> 106
aaacaggggt gtgctgggat gacac 25
<210> 107
<211> 25
<212> DNA
<213> Golden hamster
<400> 107
caccgcgggg cctcaggtgc attcc 25
<210> 108
<211> 25
<212> DNA
<213> Golden hamster
<400> 108
aaacggaatg cacctgaggc cccgc 25
<210> 109
<211> 25
<212> DNA
<213> Golden hamster
<400> 109
caccgcagga acacctcagt ctagg 25
<210> 110
<211> 25
<212> DNA
<213> Golden hamster
<400> 110
aaaccctaga ctgaggtgtt cctgc 25
<210> 111
<211> 25
<212> DNA
<213> Golden hamster
<400> 111
caccgctgtt cgccatgtca cagga 25
<210> 112
<211> 25
<212> DNA
<213> Golden hamster
<400> 112
aaactcctgt gacatggcga acagc 25
<210> 113
<211> 25
<212> DNA
<213> Golden hamster
<400> 113
caccgccagc ttggcgatct tcaca 25
<210> 114
<211> 25
<212> DNA
<213> Golden hamster
<400> 114
aaactgtgaa gatcgccaag ctggc 25
<210> 115
<211> 25
<212> DNA
<213> Golden hamster
<400> 115
caccgcagca caggttctgt ggtct 25
<210> 116
<211> 25
<212> DNA
<213> Golden hamster
<400> 116
aaacagacca cagaacctgt gctgc 25
<210> 117
<211> 25
<212> DNA
<213> Golden hamster
<400> 117
caccggacat gctcaggacg taacg 25
<210> 118
<211> 25
<212> DNA
<213> Golden hamster
<400> 118
aaaccgttac gtcctgagca tgtcc 25
<210> 119
<211> 25
<212> DNA
<213> Golden hamster
<400> 119
caccggttct acaagtgctg cactg 25
<210> 120
<211> 25
<212> DNA
<213> Golden hamster
<400> 120
aaaccagtgc agcacttgta gaacc 25
<210> 121
<211> 25
<212> DNA
<213> Golden hamster
<400> 121
caccgaccct aaagtggagc cacaa 25
<210> 122
<211> 25
<212> DNA
<213> Golden hamster
<400> 122
aaacttgtgg ctccacttta gggtc 25
<210> 123
<211> 25
<212> DNA
<213> Golden hamster
<400> 123
caccgggagg aaatcccagc cccga 25
<210> 124
<211> 25
<212> DNA
<213> Golden hamster
<400> 124
aaactcgggg ctgggatttc ctccc 25
<210> 125
<211> 25
<212> DNA
<213> Golden hamster
<400> 125
caccgcgtat agtgagttgg tggga 25
<210> 126
<211> 25
<212> DNA
<213> Golden hamster
<400> 126
aaactcccac caactcacta tacgc 25
<210> 127
<211> 25
<212> DNA
<213> Golden hamster
<400> 127
caccgtgcga cgtgacagat gagtg 25
<210> 128
<211> 25
<212> DNA
<213> Golden hamster
<400> 128
aaaccactca tctgtcacgt cgcac 25
<210> 129
<211> 25
<212> DNA
<213> Golden hamster
<400> 129
caccgttcct catccatggc caacc 25
<210> 130
<211> 25
<212> DNA
<213> Golden hamster
<400> 130
aaacggttgg ccatggatga ggaac 25
<210> 131
<211> 25
<212> DNA
<213> Golden hamster
<400> 131
caccggttct gtgtggtgac cccaa 25
<210> 132
<211> 25
<212> DNA
<213> Golden hamster
<400> 132
aaacttgggg tcaccacaca gaacc 25
<210> 133
<211> 25
<212> DNA
<213> Golden hamster
<400> 133
caccgtctaa ggacgtgtgc ttgca 25
<210> 134
<211> 25
<212> DNA
<213> Golden hamster
<400> 134
aaactgcaag cacacgtcct tagac 25
<210> 135
<211> 25
<212> DNA
<213> Golden hamster
<400> 135
caccgtcctc gggaaaggac tccct 25
<210> 136
<211> 25
<212> DNA
<213> Golden hamster
<400> 136
aaacagggag tcctttcccg aggac 25
<210> 137
<211> 25
<212> DNA
<213> Golden hamster
<400> 137
caccgccact gttctggatg tagca 25
<210> 138
<211> 25
<212> DNA
<213> Golden hamster
<400> 138
aaactgctac atccagaaca gtggc 25
<210> 139
<211> 25
<212> DNA
<213> Golden hamster
<400> 139
caccgtcctt gtgccttgac ccaag 25
<210> 140
<211> 25
<212> DNA
<213> Golden hamster
<400> 140
aaaccttggg tcaaggcaca aggac 25
<210> 141
<211> 25
<212> DNA
<213> Golden hamster
<400> 141
caccgtgatg gcactggcga gatgg 25
<210> 142
<211> 25
<212> DNA
<213> Golden hamster
<400> 142
aaacccatct cgccagtgcc atcac 25
<210> 143
<211> 25
<212> DNA
<213> Golden hamster
<400> 143
caccggacat tgagaatgag tgcct 25
<210> 144
<211> 25
<212> DNA
<213> Golden hamster
<400> 144
aaacaggcac tcattctcaa tgtcc 25
<210> 145
<211> 25
<212> DNA
<213> Golden hamster
<400> 145
caccgcatca tccttgtagt ccagc 25
<210> 146
<211> 25
<212> DNA
<213> Golden hamster
<400> 146
aaacgctgga ctacaaggat gatgc 25
<210> 147
<211> 25
<212> DNA
<213> Golden hamster
<400> 147
caccgtacct ccactctctg gcttc 25
<210> 148
<211> 25
<212> DNA
<213> Golden hamster
<400> 148
aaacgaagcc agagagtgga ggtac 25
<210> 149
<211> 25
<212> DNA
<213> Golden hamster
<400> 149
caccggaggt catggaaacg gatgg 25
<210> 150
<211> 25
<212> DNA
<213> Golden hamster
<400> 150
aaacccatcc gtttccatga cctcc 25
<210> 151
<211> 25
<212> DNA
<213> Golden hamster
<400> 151
caccggccct gatggcctta ttcca 25
<210> 152
<211> 25
<212> DNA
<213> Golden hamster
<400> 152
aaactggaat aaggccatca gggcc 25
<210> 153
<211> 25
<212> DNA
<213> Golden hamster
<400> 153
caccgtccgt ggtctgaagt ctcga 25
<210> 154
<211> 25
<212> DNA
<213> Golden hamster
<400> 154
aaactcgaga cttcagacca cggac 25
<210> 155
<211> 25
<212> DNA
<213> Golden hamster
<400> 155
caccgatcat ctctgccagc tgggt 25
<210> 156
<211> 25
<212> DNA
<213> Golden hamster
<400> 156
aaacacccag ctggcagaga tgatc 25
<210> 157
<211> 25
<212> DNA
<213> Golden hamster
<400> 157
caccgttcac tgaagcgcag gagaa 25
<210> 158
<211> 25
<212> DNA
<213> Golden hamster
<400> 158
aaacttctcc tgcgcttcag tgaac 25
<210> 159
<211> 25
<212> DNA
<213> Golden hamster
<400> 159
caccgaccca aaagcttcat cccga 25
<210> 160
<211> 25
<212> DNA
<213> Golden hamster
<400> 160
aaactcggga tgaagctttt gggtc 25
<210> 161
<211> 25
<212> DNA
<213> Golden hamster
<400> 161
caccgcaggt atcttgagaa gccca 25
<210> 162
<211> 25
<212> DNA
<213> Golden hamster
<400> 162
aaactgggct tctcaagata cctgc 25
<210> 163
<211> 25
<212> DNA
<213> Golden hamster
<400> 163
caccgctcct cagtcacaat caagg 25
<210> 164
<211> 25
<212> DNA
<213> Golden hamster
<400> 164
aaacccttga ttgtgactga ggagc 25
<210> 165
<211> 25
<212> DNA
<213> Golden hamster
<400> 165
caccgatgaa cttggtcttc aggta 25
<210> 166
<211> 25
<212> DNA
<213> Golden hamster
<400> 166
aaactacctg aagaccaagt tcatc 25
<210> 167
<211> 25
<212> DNA
<213> Golden hamster
<400> 167
caccgtttcc aactcagacg acagc 25
<210> 168
<211> 25
<212> DNA
<213> Golden hamster
<400> 168
aaacgctgtc gtctgagttg gaaac 25
<210> 169
<211> 25
<212> DNA
<213> Golden hamster
<400> 169
caccggtcca gtattgcttc aagcc 25
<210> 170
<211> 25
<212> DNA
<213> Golden hamster
<400> 170
aaacggcttg aagcaatact ggacc 25
<210> 171
<211> 25
<212> DNA
<213> Golden hamster
<400> 171
caccgcaagc gtgctgacag gcgtg 25
<210> 172
<211> 25
<212> DNA
<213> Golden hamster
<400> 172
aaaccacgcc tgtcagcacg cttgc 25
<210> 173
<211> 25
<212> DNA
<213> Golden hamster
<400> 173
caccgccacg agagatttct ccata 25
<210> 174
<211> 25
<212> DNA
<213> Golden hamster
<400> 174
aaactatgga gaaatctctc gtggc 25
<210> 175
<211> 25
<212> DNA
<213> Golden hamster
<400> 175
caccgtgatc gccgtggtgc agagt 25
<210> 176
<211> 25
<212> DNA
<213> Golden hamster
<400> 176
aaacactctg caccacggcg atcac 25
<210> 177
<211> 25
<212> DNA
<213> Golden hamster
<400> 177
caccgccgga tggagaagtc tctag 25
<210> 178
<211> 25
<212> DNA
<213> Golden hamster
<400> 178
aaacctagag acttctccat ccggc 25
<210> 179
<211> 25
<212> DNA
<213> Golden hamster
<400> 179
caccgctcaa gttcaccaca gccct 25
<210> 180
<211> 25
<212> DNA
<213> Golden hamster
<400> 180
aaacagggct gtggtgaact tgagc 25
<210> 181
<211> 25
<212> DNA
<213> Golden hamster
<400> 181
caccgtgcaa ctcagctcag atatg 25
<210> 182
<211> 25
<212> DNA
<213> Golden hamster
<400> 182
aaaccatatc tgagctgagt tgcac 25
<210> 183
<211> 25
<212> DNA
<213> Golden hamster
<400> 183
caccgcagct ggtgtccttc tgtgt 25
<210> 184
<211> 25
<212> DNA
<213> Golden hamster
<400> 184
aaacacacag aaggacacca gctgc 25
<210> 185
<211> 25
<212> DNA
<213> Golden hamster
<400> 185
caccgtacat gaatcggagc tccgg 25
<210> 186
<211> 25
<212> DNA
<213> Golden hamster
<400> 186
aaacccggag ctccgattca tgtac 25
<210> 187
<211> 25
<212> DNA
<213> Golden hamster
<400> 187
caccggtgta ggaactccca tactc 25
<210> 188
<211> 25
<212> DNA
<213> Golden hamster
<400> 188
aaacgagtat gggagttcct acacc 25
<210> 189
<211> 25
<212> DNA
<213> Golden hamster
<400> 189
caccgtcaca gaccagccag atggt 25
<210> 190
<211> 25
<212> DNA
<213> Golden hamster
<400> 190
aaacaccatc tggctggtct gtgac 25
<210> 191
<211> 25
<212> DNA
<213> Golden hamster
<400> 191
caccgggcat ggacttctgc cagga 25
<210> 192
<211> 25
<212> DNA
<213> Golden hamster
<400> 192
aaactcctgg cagaagtcca tgccc 25
<210> 193
<211> 25
<212> DNA
<213> Golden hamster
<400> 193
caccgttccc tgtgcagtga cccac 25
<210> 194
<211> 25
<212> DNA
<213> Golden hamster
<400> 194
aaacgtgggt cactgcacag ggaac 25
<210> 195
<211> 25
<212> DNA
<213> Golden hamster
<400> 195
caccgatctc gctcattcct gaggc 25
<210> 196
<211> 25
<212> DNA
<213> Golden hamster
<400> 196
aaacgcctca ggaatgagcg agatc 25
<210> 197
<211> 25
<212> DNA
<213> Golden hamster
<400> 197
caccgcaaga tgctgtcaaa gttta 25
<210> 198
<211> 25
<212> DNA
<213> Golden hamster
<400> 198
aaactaaact ttgacagcat cttgc 25
<210> 199
<211> 25
<212> DNA
<213> Golden hamster
<400> 199
caccggtggc ttctcagtcc acatg 25
<210> 200
<211> 25
<212> DNA
<213> Golden hamster
<400> 200
aaaccatgtg gactgagaag ccacc 25
<210> 201
<211> 25
<212> DNA
<213> Golden hamster
<400> 201
caccgcaaga tgctgtcaaa gttta 25
<210> 202
<211> 25
<212> DNA
<213> Golden hamster
<400> 202
aaactaaact ttgacagcat cttgc 25
<210> 203
<211> 25
<212> DNA
<213> Golden hamster
<400> 203
caccgaaatg gctctcttct tagat 25
<210> 204
<211> 25
<212> DNA
<213> Golden hamster
<400> 204
aaacatctaa gaagagagcc atttc 25
<210> 205
<211> 25
<212> DNA
<213> Golden hamster
<400> 205
caccgaagct acggcccgca aaggg 25
<210> 206
<211> 25
<212> DNA
<213> Golden hamster
<400> 206
aaaccccttt gcgggccgta gcttc 25
<210> 207
<211> 25
<212> DNA
<213> Golden hamster
<400> 207
caccgcctcc tgcggctagg acctc 25
<210> 208
<211> 25
<212> DNA
<213> Golden hamster
<400> 208
aaacgaggtc ctagccgcag gaggc 25
<210> 209
<211> 25
<212> DNA
<213> Golden hamster
<400> 209
caccgtatcg ttaccaggtg ccctc 25
<210> 210
<211> 25
<212> DNA
<213> Golden hamster
<400> 210
aaacgagggc acctggtaac gatac 25
<210> 211
<211> 25
<212> DNA
<213> Golden hamster
<400> 211
caccggcact tgaatgccta ctacg 25
<210> 212
<211> 25
<212> DNA
<213> Golden hamster
<400> 212
aaaccgtagt aggcattcaa gtgcc 25
<210> 213
<211> 25
<212> DNA
<213> Golden hamster
<400> 213
caccgtattg tcaccagatg ccctc 25
<210> 214
<211> 25
<212> DNA
<213> Golden hamster
<400> 214
aaacgagggc atctggtgac aatac 25
<210> 215
<211> 25
<212> DNA
<213> Golden hamster
<400> 215
caccgtagcc tgaggtcttc aagtt 25
<210> 216
<211> 25
<212> DNA
<213> Golden hamster
<400> 216
aaacaacttg aagacctcag gctac 25
<210> 217
<211> 25
<212> DNA
<213> Golden hamster
<400> 217
caccgactgt tctggaggag atctc 25
<210> 218
<211> 25
<212> DNA
<213> Golden hamster
<400> 218
aaacgagatc tcctccagaa cagtc 25
<210> 219
<211> 25
<212> DNA
<213> Golden hamster
<400> 219
caccgctttc cctgcataaa catga 25
<210> 220
<211> 25
<212> DNA
<213> Golden hamster
<400> 220
aaactcatgt ttatgcaggg aaagc 25
<210> 221
<211> 25
<212> DNA
<213> Golden hamster
<400> 221
caccgtcctc cttggcagta ctcca 25
<210> 222
<211> 25
<212> DNA
<213> Golden hamster
<400> 222
aaactggagt actgccaagg aggac 25
<210> 223
<211> 25
<212> DNA
<213> Golden hamster
<400> 223
caccggtcac aggcactgca cggct 25
<210> 224
<211> 25
<212> DNA
<213> Golden hamster
<400> 224
aaacagccgt gcagtgcctg tgacc 25
<210> 225
<211> 25
<212> DNA
<213> Golden hamster
<400> 225
caccggatgc gagcagttag tgagc 25
<210> 226
<211> 25
<212> DNA
<213> Golden hamster
<400> 226
aaacgctcac taactgctcg catcc 25
<210> 227
<211> 25
<212> DNA
<213> Golden hamster
<400> 227
caccgaggcg cctgatactg gcatt 25
<210> 228
<211> 25
<212> DNA
<213> Golden hamster
<400> 228
aaacaatgcc agtatcaggc gcctc 25
<210> 229
<211> 25
<212> DNA
<213> Golden hamster
<400> 229
caccgtcaac tcagccagct accgg 25
<210> 230
<211> 25
<212> DNA
<213> Golden hamster
<400> 230
aaacccggta gctggctgag ttgac 25
<210> 231
<211> 25
<212> DNA
<213> Golden hamster
<400> 231
caccgtgcta caggcagtca ccgat 25
<210> 232
<211> 25
<212> DNA
<213> Golden hamster
<400> 232
aaacatcggt gactgcctgt agcac 25
<210> 233
<211> 25
<212> DNA
<213> Golden hamster
<400> 233
caccgagatc atcagaagaa atatg 25
<210> 234
<211> 25
<212> DNA
<213> Golden hamster
<400> 234
aaaccatatt tcttctgatg atctc 25
<210> 235
<211> 25
<212> DNA
<213> Golden hamster
<400> 235
caccgtctcc gtgatgcaat ttaac 25
<210> 236
<211> 25
<212> DNA
<213> Golden hamster
<400> 236
aaacgttaaa ttgcatcacg gagac 25
<210> 237
<211> 25
<212> DNA
<213> Golden hamster
<400> 237
caccgtaaga gcttcagccc acacg 25
<210> 238
<211> 25
<212> DNA
<213> Golden hamster
<400> 238
aaaccgtgtg ggctgaagct cttac 25
<210> 239
<211> 25
<212> DNA
<213> Golden hamster
<400> 239
caccggcgtt ggctgcaggt gactc 25
<210> 240
<211> 25
<212> DNA
<213> Golden hamster
<400> 240
aaacgagtca cctgcagcca acgcc 25
<210> 241
<211> 25
<212> DNA
<213> Golden hamster
<400> 241
caccgatcag cctcaaccac agttg 25
<210> 242
<211> 25
<212> DNA
<213> Golden hamster
<400> 242
aaaccaactg tggttgaggc tgatc 25
<210> 243
<211> 25
<212> DNA
<213> Golden hamster
<400> 243
caccgtcatc tcccctgtgt tgtca 25
<210> 244
<211> 25
<212> DNA
<213> Golden hamster
<400> 244
aaactgacaa cacaggggag atgac 25
<210> 245
<211> 25
<212> DNA
<213> Golden hamster
<400> 245
caccggagtt ccagactctc aagga 25
<210> 246
<211> 25
<212> DNA
<213> Golden hamster
<400> 246
aaactccttg agagtctgga actcc 25
<210> 247
<211> 25
<212> DNA
<213> Golden hamster
<400> 247
caccggagcc acgaagggct gctct 25
<210> 248
<211> 25
<212> DNA
<213> Golden hamster
<400> 248
aaacagagca gcccttcgtg gctcc 25
<210> 249
<211> 25
<212> DNA
<213> Golden hamster
<400> 249
caccggaaca cttcacgctg ctcac 25
<210> 250
<211> 25
<212> DNA
<213> Golden hamster
<400> 250
aaacgtgagc agcgtgaagt gttcc 25
<210> 251
<211> 25
<212> DNA
<213> Golden hamster
<400> 251
caccgttgga gtccttctgg aggcc 25
<210> 252
<211> 25
<212> DNA
<213> Golden hamster
<400> 252
aaacggcctc cagaaggact ccaac 25
<210> 253
<211> 25
<212> DNA
<213> Golden hamster
<400> 253
caccggataa ttgggcacgg tgccc 25
<210> 254
<211> 25
<212> DNA
<213> Golden hamster
<400> 254
aaacgggcac cgtgcccaat tatcc 25
<210> 255
<211> 25
<212> DNA
<213> Golden hamster
<400> 255
caccgatcaa atgctctggg gcccc 25
<210> 256
<211> 25
<212> DNA
<213> Golden hamster
<400> 256
aaacggggcc ccagagcatt tgatc 25
<210> 257
<211> 25
<212> DNA
<213> Golden hamster
<400> 257
caccgtagtg gaggcgtttg tccgc 25
<210> 258
<211> 25
<212> DNA
<213> Golden hamster
<400> 258
aaacgcggac aaacgcctcc actac 25
<210> 259
<211> 25
<212> DNA
<213> Golden hamster
<400> 259
caccgagatg atcgttcctc cttcg 25
<210> 260
<211> 25
<212> DNA
<213> Golden hamster
<400> 260
aaaccgaagg aggaacgatc atctc 25
<210> 261
<211> 25
<212> DNA
<213> Golden hamster
<400> 261
caccgtgcaa gccaccctcc acaag 25
<210> 262
<211> 25
<212> DNA
<213> Golden hamster
<400> 262
aaaccttgtg gagggtggct tgcac 25
<210> 263
<211> 25
<212> DNA
<213> Golden hamster
<400> 263
caccggcgga agtggaaaaa gaact 25
<210> 264
<211> 25
<212> DNA
<213> Golden hamster
<400> 264
aaacagttct ttttccactt ccgcc 25
<210> 265
<211> 25
<212> DNA
<213> Golden hamster
<400> 265
caccggagcc tctttgtgtt tggtc 25
<210> 266
<211> 25
<212> DNA
<213> Golden hamster
<400> 266
aaacgaccaa acacaaagag gctcc 25
<210> 267
<211> 25
<212> DNA
<213> Golden hamster
<400> 267
caccgaacag aataacaagc gaggg 25
<210> 268
<211> 25
<212> DNA
<213> Golden hamster
<400> 268
aaacccctcg cttgttattc tgttc 25
<210> 269
<211> 25
<212> DNA
<213> Golden hamster
<400> 269
caccgaagta ctgaggaagc gcgac 25
<210> 270
<211> 25
<212> DNA
<213> Golden hamster
<400> 270
aaacgtcgcg cttcctcagt acttc 25
<210> 271
<211> 25
<212> DNA
<213> Golden hamster
<400> 271
caccgcgtgc ccaaaatgtt gcgcc 25
<210> 272
<211> 25
<212> DNA
<213> Golden hamster
<400> 272
aaacggcgca acattttggg cacgc 25
<210> 273
<211> 25
<212> DNA
<213> Golden hamster
<400> 273
caccgaacaa tggcattagc taccg 25
<210> 274
<211> 25
<212> DNA
<213> Golden hamster
<400> 274
aaaccggtag ctaatgccat tgttc 25
<210> 275
<211> 25
<212> DNA
<213> Golden hamster
<400> 275
caccgggtcc ttcctcttct ttaga 25
<210> 276
<211> 25
<212> DNA
<213> Golden hamster
<400> 276
aaactctaaa gaagaggaag gaccc 25
<210> 277
<211> 25
<212> DNA
<213> Golden hamster
<400> 277
caccggttaa atgggccaca cacct 25
<210> 278
<211> 25
<212> DNA
<213> Golden hamster
<400> 278
aaacaggtgt gtggcccatt taacc 25
<210> 279
<211> 25
<212> DNA
<213> Golden hamster
<400> 279
caccgcaccc taaacagcag caagg 25
<210> 280
<211> 25
<212> DNA
<213> Golden hamster
<400> 280
aaacccttgc tgctgtttag ggtgc 25
<210> 281
<211> 25
<212> DNA
<213> Golden hamster
<400> 281
caccgtcccc gcgagaacaa gacag 25
<210> 282
<211> 25
<212> DNA
<213> Golden hamster
<400> 282
aaacctgtct tgttctcgcg gggac 25
<210> 283
<211> 25
<212> DNA
<213> Golden hamster
<400> 283
caccggacat ctttccctca agttc 25
<210> 284
<211> 25
<212> DNA
<213> Golden hamster
<400> 284
aaacgaactt gagggaaaga tgtcc 25
<210> 285
<211> 25
<212> DNA
<213> Golden hamster
<400> 285
caccgaatgg attctggaat atatc 25
<210> 286
<211> 25
<212> DNA
<213> Golden hamster
<400> 286
aaacgatata ttccagaatc cattc 25
<210> 287
<211> 25
<212> DNA
<213> Golden hamster
<400> 287
caccgtactc gagttaaccg gaagg 25
<210> 288
<211> 25
<212> DNA
<213> Golden hamster
<400> 288
aaacccttcc ggttaactcg agtac 25
<210> 289
<211> 25
<212> DNA
<213> Golden hamster
<400> 289
caccgtcgtt agaatggacg attcc 25
<210> 290
<211> 25
<212> DNA
<213> Golden hamster
<400> 290
aaacggaatc gtccattcta acgac 25
<210> 291
<211> 25
<212> DNA
<213> Golden hamster
<400> 291
caccggagag caagacgtgt acgca 25
<210> 292
<211> 25
<212> DNA
<213> Golden hamster
<400> 292
aaactgcgta cacgtcttgc tctcc 25
<210> 293
<211> 25
<212> DNA
<213> Golden hamster
<400> 293
caccgtgtcc gaagtcctgt gctct 25
<210> 294
<211> 25
<212> DNA
<213> Golden hamster
<400> 294
aaacagagca caggacttcg gacac 25
<210> 295
<211> 25
<212> DNA
<213> Golden hamster
<400> 295
caccgctgcc agaactgatg tccac 25
<210> 296
<211> 25
<212> DNA
<213> Golden hamster
<400> 296
aaacgtggac atcagttctg gcagc 25
<210> 297
<211> 25
<212> DNA
<213> Golden hamster
<400> 297
caccgttctt cattcaggcc tgtgg 25
<210> 298
<211> 25
<212> DNA
<213> Golden hamster
<400> 298
aaacccacag gcctgaatga agaac 25
<210> 299
<211> 25
<212> DNA
<213> Golden hamster
<400> 299
caccggtgcc ccgtcaagac tgccc 25
<210> 300
<211> 25
<212> DNA
<213> Golden hamster
<400> 300
aaacgggcag tcttgacggg gcacc 25
<210> 301
<211> 25
<212> DNA
<213> Golden hamster
<400> 301
caccgacaga gggcctgcac aaacc 25
<210> 302
<211> 25
<212> DNA
<213> Golden hamster
<400> 302
aaacggtttg tgcaggccct ctgtc 25
<210> 303
<211> 25
<212> DNA
<213> Golden hamster
<400> 303
caccgactcc tgctcaaaat tcacg 25
<210> 304
<211> 25
<212> DNA
<213> Golden hamster
<400> 304
aaaccgtgaa ttttgagcag gagtc 25
<210> 305
<211> 25
<212> DNA
<213> Golden hamster
<400> 305
caccgatgtg ttactcggtg gccga 25
<210> 306
<211> 25
<212> DNA
<213> Golden hamster
<400> 306
aaactcggcc accgagtaac acatc 25
<210> 307
<211> 25
<212> DNA
<213> Golden hamster
<400> 307
caccggttgg tggctcccgc ggcaa 25
<210> 308
<211> 25
<212> DNA
<213> Golden hamster
<400> 308
aaacttgccg cgggagccac caacc 25
<210> 309
<211> 25
<212> DNA
<213> Golden hamster
<400> 309
caccgcactg ccatgcgcca cccag 25
<210> 310
<211> 25
<212> DNA
<213> Golden hamster
<400> 310
aaacctgggt ggcgcatggc agtgc 25
<210> 311
<211> 25
<212> DNA
<213> Golden hamster
<400> 311
caccgaaaat cacagctact acttg 25
<210> 312
<211> 25
<212> DNA
<213> Golden hamster
<400> 312
aaaccaagta gtagctgtga ttttc 25
<210> 313
<211> 25
<212> DNA
<213> Golden hamster
<400> 313
caccggcatc tgttgttaca ccaag 25
<210> 314
<211> 25
<212> DNA
<213> Golden hamster
<400> 314
aaaccttggt gtaacaacag atgcc 25
<210> 315
<211> 25
<212> DNA
<213> Golden hamster
<400> 315
caccgtctat tgaagaggtg aaatt 25
<210> 316
<211> 25
<212> DNA
<213> Golden hamster
<400> 316
aaacaatttc acctcttcaa tagac 25
<210> 317
<211> 25
<212> DNA
<213> Golden hamster
<400> 317
caccggagtc ctatacctga ggacg 25
<210> 318
<211> 25
<212> DNA
<213> Golden hamster
<400> 318
aaaccgtcct caggtatagg actcc 25
<210> 319
<211> 25
<212> DNA
<213> Golden hamster
<400> 319
caccgctgcc cacagtgtag aattc 25
<210> 320
<211> 25
<212> DNA
<213> Golden hamster
<400> 320
aaacgaattc tacactgtgg gcagc 25
<210> 321
<211> 25
<212> DNA
<213> Golden hamster
<400> 321
caccggagcc agacgaggaa ctggc 25
<210> 322
<211> 25
<212> DNA
<213> Golden hamster
<400> 322
aaacgccagt tcctcgtctg gctcc 25
<210> 323
<211> 25
<212> DNA
<213> Golden hamster
<400> 323
caccgcagat ctactgtcgg aatga 25
<210> 324
<211> 25
<212> DNA
<213> Golden hamster
<400> 324
aaactcattc cgacagtaga tctgc 25
<210> 325
<211> 25
<212> DNA
<213> Golden hamster
<400> 325
caccggggca ggacaccacc acaca 25
<210> 326
<211> 25
<212> DNA
<213> Golden hamster
<400> 326
aaactgtgtg gtggtgtcct gcccc 25
<210> 327
<211> 25
<212> DNA
<213> Golden hamster
<400> 327
caccgacgct catgctgatg gatca 25
<210> 328
<211> 25
<212> DNA
<213> Golden hamster
<400> 328
aaactgatcc atcagcatga gcgtc 25
<210> 329
<211> 25
<212> DNA
<213> Golden hamster
<400> 329
caccgcatcc tgcggaggcc tttcc 25
<210> 330
<211> 25
<212> DNA
<213> Golden hamster
<400> 330
aaacggaaag gcctccgcag gatgc 25
<210> 331
<211> 25
<212> DNA
<213> Golden hamster
<400> 331
caccgacttg ctgcatgatc taaca 25
<210> 332
<211> 25
<212> DNA
<213> Golden hamster
<400> 332
aaactgttag atcatgcagc aagtc 25
<210> 333
<211> 25
<212> DNA
<213> Golden hamster
<400> 333
caccgccaat gatgccctct tgcgg 25
<210> 334
<211> 25
<212> DNA
<213> Golden hamster
<400> 334
aaacccgcaa gagggcatca ttggc 25
<210> 335
<211> 25
<212> DNA
<213> Golden hamster
<400> 335
caccgaggtg tcggctgttc cttca 25
<210> 336
<211> 25
<212> DNA
<213> Golden hamster
<400> 336
aaactgaagg aacagccgac acctc 25
<210> 337
<211> 25
<212> DNA
<213> Golden hamster
<400> 337
caccgagtat gacgctctcc tgcct 25
<210> 338
<211> 25
<212> DNA
<213> Golden hamster
<400> 338
aaacaggcag gagagcgtca tactc 25
<210> 339
<211> 25
<212> DNA
<213> Golden hamster
<400> 339
caccggccat cagtaccagt gccca 25
<210> 340
<211> 25
<212> DNA
<213> Golden hamster
<400> 340
aaactgggca ctggtactga tggcc 25
<210> 341
<211> 25
<212> DNA
<213> Golden hamster
<400> 341
caccgaggct ggactgaagc actcc 25
<210> 342
<211> 25
<212> DNA
<213> Golden hamster
<400> 342
aaacggagtg cttcagtcca gcctc 25
<210> 343
<211> 25
<212> DNA
<213> Golden hamster
<400> 343
caccgcgaat gccccatctg cttga 25
<210> 344
<211> 25
<212> DNA
<213> Golden hamster
<400> 344
aaactcaagc agatggggca ttcgc 25
<210> 345
<211> 25
<212> DNA
<213> Golden hamster
<400> 345
caccgcaaaa acactgaatg agcaa 25
<210> 346
<211> 25
<212> DNA
<213> Golden hamster
<400> 346
aaacttgctc attcagtgtt tttgc 25
<210> 347
<211> 25
<212> DNA
<213> Golden hamster
<400> 347
caccgatgga agcacaacag tgtaa 25
<210> 348
<211> 25
<212> DNA
<213> Golden hamster
<400> 348
aaacttacac tgttgtgctt ccatc 25
<210> 349
<211> 25
<212> DNA
<213> Golden hamster
<400> 349
caccgcaagc tccgaggccg aggtc 25
<210> 350
<211> 25
<212> DNA
<213> Golden hamster
<400> 350
aaacgacctc ggcctcggag cttgc 25
<210> 351
<211> 25
<212> DNA
<213> Golden hamster
<400> 351
caccgaatgt ccagtcgttg aggcc 25
<210> 352
<211> 25
<212> DNA
<213> Golden hamster
<400> 352
aaacggcctc aacgactgga cattc 25
<210> 353
<211> 25
<212> DNA
<213> Golden hamster
<400> 353
caccgtccag agtgggggtg aacca 25
<210> 354
<211> 25
<212> DNA
<213> Golden hamster
<400> 354
aaactggttc acccccactc tggac 25
<210> 355
<211> 25
<212> DNA
<213> Golden hamster
<400> 355
caccgtactt ctggaagaca cctca 25
<210> 356
<211> 25
<212> DNA
<213> Golden hamster
<400> 356
aaactgaggt gtcttccaga agtac 25
<210> 357
<211> 25
<212> DNA
<213> Golden hamster
<400> 357
caccgtcctg aaaacttcat caggg 25
<210> 358
<211> 25
<212> DNA
<213> Golden hamster
<400> 358
aaacccctga tgaagttttc aggac 25
<210> 359
<211> 25
<212> DNA
<213> Golden hamster
<400> 359
caccgcctca gaaaccctgc tccaa 25
<210> 360
<211> 25
<212> DNA
<213> Golden hamster
<400> 360
aaacttggag cagggtttct gaggc 25
<210> 361
<211> 25
<212> DNA
<213> Golden hamster
<400> 361
caccgatggt gatgcaccaa aggat 25
<210> 362
<211> 25
<212> DNA
<213> Golden hamster
<400> 362
aaacatcctt tggtgcatca ccatc 25
<210> 363
<211> 25
<212> DNA
<213> Golden hamster
<400> 363
caccgtacgc cgattcggat gtgcc 25
<210> 364
<211> 25
<212> DNA
<213> Golden hamster
<400> 364
aaacggcaca tccgaatcgg cgtac 25
<210> 365
<211> 25
<212> DNA
<213> Golden hamster
<400> 365
caccggatca cgttgggctc ctctt 25
<210> 366
<211> 25
<212> DNA
<213> Golden hamster
<400> 366
aaacaagagg agcccaacgt gatcc 25
<210> 367
<211> 25
<212> DNA
<213> Golden hamster
<400> 367
caccgttcta gagaagctgc aacgg 25
<210> 368
<211> 25
<212> DNA
<213> Golden hamster
<400> 368
aaacccgttg cagcttctct agaac 25
<210> 369
<211> 25
<212> DNA
<213> Golden hamster
<400> 369
caccgatgga accccaccac tttct 25
<210> 370
<211> 25
<212> DNA
<213> Golden hamster
<400> 370
aaacagaaag tggtggggtt ccatc 25
<210> 371
<211> 25
<212> DNA
<213> Golden hamster
<400> 371
caccgacggt cacctgcgtc cggtc 25
<210> 372
<211> 25
<212> DNA
<213> Golden hamster
<400> 372
aaacgaccgg acgcaggtga ccgtc 25
<210> 373
<211> 25
<212> DNA
<213> Golden hamster
<400> 373
caccgtggag agtgatagaa ctcag 25
<210> 374
<211> 25
<212> DNA
<213> Golden hamster
<400> 374
aaacctgagt tctatcactc tccac 25
<210> 375
<211> 25
<212> DNA
<213> Golden hamster
<400> 375
caccgaaact ctggcaaaac cacaa 25
<210> 376
<211> 25
<212> DNA
<213> Golden hamster
<400> 376
aaacttgtgg ttttgccaga gtttc 25
<210> 377
<211> 25
<212> DNA
<213> Golden hamster
<400> 377
caccgataga ttcttgaatg atatt 25
<210> 378
<211> 25
<212> DNA
<213> Golden hamster
<400> 378
aaacaatatc attcaagaat ctatc 25
<210> 379
<211> 25
<212> DNA
<213> Golden hamster
<400> 379
caccgagtct gtccagaaat ttagc 25
<210> 380
<211> 25
<212> DNA
<213> Golden hamster
<400> 380
aaacgctaaa tttctggaca gactc 25
<210> 381
<211> 25
<212> DNA
<213> Golden hamster
<400> 381
caccgggtag cttaagcttt cacac 25
<210> 382
<211> 25
<212> DNA
<213> Golden hamster
<400> 382
aaacgtgtga aagcttaagc taccc 25
<210> 383
<211> 25
<212> DNA
<213> Golden hamster
<400> 383
caccgtcctc caagcctacc gcgag 25
<210> 384
<211> 25
<212> DNA
<213> Golden hamster
<400> 384
aaacctcgcg gtaggcttgg aggac 25
<210> 385
<211> 25
<212> DNA
<213> Golden hamster
<400> 385
caccgagcgt agctgccgag cgctg 25
<210> 386
<211> 25
<212> DNA
<213> Golden hamster
<400> 386
aaaccagcgc tcggcagcta cgctc 25
<210> 387
<211> 25
<212> DNA
<213> Golden hamster
<400> 387
caccgcagcg ttgaagatga gtgga 25
<210> 388
<211> 25
<212> DNA
<213> Golden hamster
<400> 388
aaactccact catcttcaac gctgc 25
<210> 389
<211> 25
<212> DNA
<213> Golden hamster
<400> 389
caccgcaggc cagcaactga caatc 25
<210> 390
<211> 25
<212> DNA
<213> Golden hamster
<400> 390
aaacgattgt cagttgctgg cctgc 25
<210> 391
<211> 25
<212> DNA
<213> Golden hamster
<400> 391
caccgtctgc tggattgtgt gcaca 25
<210> 392
<211> 25
<212> DNA
<213> Golden hamster
<400> 392
aaactgtgca cacaatccag cagac 25
<210> 393
<211> 25
<212> DNA
<213> Golden hamster
<400> 393
caccgccagg agagaacctc ctatt 25
<210> 394
<211> 25
<212> DNA
<213> Golden hamster
<400> 394
aaacaatagg aggttctctc ctggc 25
<210> 395
<211> 25
<212> DNA
<213> Golden hamster
<400> 395
caccggaatg ttacagcctg gatgc 25
<210> 396
<211> 25
<212> DNA
<213> Golden hamster
<400> 396
aaacgcatcc aggctgtaac attcc 25
<210> 397
<211> 25
<212> DNA
<213> Golden hamster
<400> 397
caccgcctca catctgcgtg ctgtc 25
<210> 398
<211> 25
<212> DNA
<213> Golden hamster
<400> 398
aaacgacagc acgcagatgt gaggc 25
<210> 399
<211> 25
<212> DNA
<213> Golden hamster
<400> 399
caccgcaact gatcaaaact ctctg 25
<210> 400
<211> 25
<212> DNA
<213> Golden hamster
<400> 400
aaaccagaga gttttgatca gttgc 25
<210> 401
<211> 25
<212> DNA
<213> Golden hamster
<400> 401
caccgtggca gcggatcaag tttgt 25
<210> 402
<211> 25
<212> DNA
<213> Golden hamster
<400> 402
aaacacaaac ttgatccgct gccac 25
<210> 403
<211> 25
<212> DNA
<213> Golden hamster
<400> 403
caccggaccg tagtgctgca gggcc 25
<210> 404
<211> 25
<212> DNA
<213> Golden hamster
<400> 404
aaacggccct gcagcactac ggtcc 25
<210> 405
<211> 25
<212> DNA
<213> Golden hamster
<400> 405
caccggaccc tttcgaggct actag 25
<210> 406
<211> 25
<212> DNA
<213> Golden hamster
<400> 406
aaacctagta gcctcgaaag ggtcc 25
<210> 407
<211> 25
<212> DNA
<213> Golden hamster
<400> 407
caccgcatcc tcttgtgtct catac 25
<210> 408
<211> 25
<212> DNA
<213> Golden hamster
<400> 408
aaacgtatga gacacaagag gatgc 25
<210> 409
<211> 25
<212> DNA
<213> Golden hamster
<400> 409
caccgtactg agtgaaggat tggct 25
<210> 410
<211> 25
<212> DNA
<213> Golden hamster
<400> 410
aaacagccaa tccttcactc agtac 25
<210> 411
<211> 25
<212> DNA
<213> Golden hamster
<400> 411
caccgaggat atcgtatctg atcct 25
<210> 412
<211> 25
<212> DNA
<213> Golden hamster
<400> 412
aaacaggatc agatacgata tcctc 25
<210> 413
<211> 25
<212> DNA
<213> Golden hamster
<400> 413
caccggctat ccatcaagac agaac 25
<210> 414
<211> 25
<212> DNA
<213> Golden hamster
<400> 414
aaacgttctg tcttgatgga tagcc 25
<210> 415
<211> 25
<212> DNA
<213> Golden hamster
<400> 415
caccgattag tgccatagag gttca 25
<210> 416
<211> 25
<212> DNA
<213> Golden hamster
<400> 416
aaactgaacc tctatggcac taatc 25
<210> 417
<211> 25
<212> DNA
<213> Golden hamster
<400> 417
caccgtctga cttcagactg ctgta 25
<210> 418
<211> 25
<212> DNA
<213> Golden hamster
<400> 418
aaactacagc agtctgaagt cagac 25
<210> 419
<211> 25
<212> DNA
<213> Golden hamster
<400> 419
caccgccagg agggcattgg cacga 25
<210> 420
<211> 25
<212> DNA
<213> Golden hamster
<400> 420
aaactcgtgc caatgccctc ctggc 25
<210> 421
<211> 25
<212> DNA
<213> Golden hamster
<400> 421
caccgtctgc aaagtctaag tattt 25
<210> 422
<211> 25
<212> DNA
<213> Golden hamster
<400> 422
aaacaaatac ttagactttg cagac 25
<210> 423
<211> 25
<212> DNA
<213> Golden hamster
<400> 423
caccgtatgc cttccctgtg cagaa 25
<210> 424
<211> 25
<212> DNA
<213> Golden hamster
<400> 424
aaacttctgc acagggaagg catac 25
<210> 425
<211> 25
<212> DNA
<213> Golden hamster
<400> 425
caccgaaagt attggaaaag acgtc 25
<210> 426
<211> 25
<212> DNA
<213> Golden hamster
<400> 426
aaacgacgtc ttttccaata ctttc 25
<210> 427
<211> 25
<212> DNA
<213> Golden hamster
<400> 427
caccgatcgg caacagggtc cctcg 25
<210> 428
<211> 25
<212> DNA
<213> Golden hamster
<400> 428
aaaccgaggg accctgttgc cgatc 25
<210> 429
<211> 25
<212> DNA
<213> Golden hamster
<400> 429
caccgctctg gcaatgagtg cttgc 25
<210> 430
<211> 25
<212> DNA
<213> Golden hamster
<400> 430
aaacgcaagc actcattgcc agagc 25
<210> 431
<211> 25
<212> DNA
<213> Golden hamster
<400> 431
caccgctgca gtgcagctgt cgaat 25
<210> 432
<211> 25
<212> DNA
<213> Golden hamster
<400> 432
aaacattcga cagctgcact gcagc 25
<210> 433
<211> 25
<212> DNA
<213> Golden hamster
<400> 433
caccgcacct ttcatgacac aggac 25
<210> 434
<211> 25
<212> DNA
<213> Golden hamster
<400> 434
aaacgtcctg tgtcatgaaa ggtgc 25
<210> 435
<211> 25
<212> DNA
<213> Golden hamster
<400> 435
caccggtcat atcctcaaac accgg 25
<210> 436
<211> 25
<212> DNA
<213> Golden hamster
<400> 436
aaacccggtg tttgaggata tgacc 25
<210> 437
<211> 25
<212> DNA
<213> Golden hamster
<400> 437
caccgaattg tatcttgttg tgtcc 25
<210> 438
<211> 25
<212> DNA
<213> Golden hamster
<400> 438
aaacggacac aacaagatac aattc 25
<210> 439
<211> 25
<212> DNA
<213> Golden hamster
<400> 439
caccgcctgc cagggaaaag gcgcc 25
<210> 440
<211> 25
<212> DNA
<213> Golden hamster
<400> 440
aaacggcgcc ttttccctgg caggc 25
<210> 441
<211> 25
<212> DNA
<213> Golden hamster
<400> 441
caccgatgaa agaggtgatg ctgtg 25
<210> 442
<211> 25
<212> DNA
<213> Golden hamster
<400> 442
aaaccacagc atcacctctt tcatc 25
<210> 443
<211> 25
<212> DNA
<213> Golden hamster
<400> 443
caccgctcct ccaggggcag cacga 25
<210> 444
<211> 25
<212> DNA
<213> Golden hamster
<400> 444
aaactcgtgc tgcccctgga ggagc 25
<210> 445
<211> 25
<212> DNA
<213> Golden hamster
<400> 445
caccgttcca gtgcctccta attga 25
<210> 446
<211> 25
<212> DNA
<213> Golden hamster
<400> 446
aaactcaatt aggaggcact ggaac 25

Claims (3)

1. A method of enhancing viral proliferation, comprising:
firstly, analyzing genome sequences of golden hamster, aiming at respective gene sequences of recognition receptor protein, effector protein, stress protein, protein kinase, interferon and downstream regulation product thereof of innate immune system, selecting target sequences according to sequence recognition requirement of gene editing in CRISPR/Cas9 system; selecting at least two target sequences from each gene sequence; aiming at each target sequence, designing a pair of DNA oligo primers, wherein the primers are sequence 1-446;
annealing each pair of target sequence primers to generate a DNA double strand with a sticky end; treating a gRNA expression vector skeleton by using restriction endonuclease to generate a cohesive end which is the same as a primer annealing product, and performing T4DNA ligase action to obtain a pUC-gRNA-library for a subsequent transfection experiment;
transfecting BHK21 cells by using a pUC-gRNA-library;
carrying out cell sorting of a flow cytometer on the transfected BHK21 cells, and sorting a cell population with green fluorescence into a pore plate; after the cell growth is recovered, establishing a library and storing the library as a BHK21-KO-IM cell population;
sorting the BHK21-KO-IM cell population into a 96-well plate by a flow cytometer for culture, wherein each well is provided with one cell clone; after the growth is recovered, adjusting the cell density of each hole to be approximately the same, and manufacturing a compound plate for comparing the virus proliferation efficiency after the cells are amplified;
removing culture supernatant, and respectively inoculating virus proliferation maintenance solutions containing the same components; observing cytopathic effect every day, and measuring the titer of each virus multiplication sample; if the cell pathological changes exist, the cell pathological changes are judged to be positive, otherwise, the cell pathological changes are negative, the virus multiplication capacities of different cell clones are compared, and the cell clones with the improved multiplication capacity are obtained.
2. A method of enhancing viral proliferation, comprising:
firstly, analyzing genome sequences of golden hamster, aiming at respective gene sequences of recognition receptor protein, effector protein, stress protein, protein kinase, interferon and downstream regulation product thereof of innate immune system, selecting target sequences according to sequence recognition requirement of gene editing in CRISPR/Cas9 system; selecting at least two target sequences from each gene sequence; aiming at each target sequence, designing a pair of DNA oligo primers, wherein the primers are sequence 1-446;
annealing each pair of target sequence primers to generate a DNA double strand with a sticky end; treating a gRNA expression vector skeleton by using restriction endonuclease to generate a cohesive end which is the same as a primer annealing product, and performing T4DNA ligase action to obtain a pUC-gRNA-library for a subsequent transfection experiment;
initial cell density of 1X 10 for seeding6BHK21 cells/ml were cultured overnight in 6-well plates and incubated for 10 min with the pre-transfection medium replaced with Opti-MEM medium; adding 100ul of Polyethyleneimine (PEI) reagent into 1ml of Opti-MEM, uniformly mixing, adding 1-10 ug of pCas9-IRES-GFP and pUC-gRNA-library plasmid vector into 1ml of Opti-MEM, uniformly mixing, and incubating for 5 minutes at room temperature; after incubation is finished, uniformly mixing the two mixed solutions in a dropwise adding mode, and incubating for 15 minutes at room temperature to form a PEI-DNA transfection complex; adding 2ml of the transfection complex into BHK21 cells in a 6-well plate in a dropwise adding mode for transfection; 8 hours after transfection, the transfection complex was removed and replaced withThe normal medium of the BHK21 cells was cultured for 24 hours;
BHK21 cells 24 hours after transfection are used for cell sorting of a flow cytometer after digestion, dispersion, centrifugal recovery and PBS washing; before sorting, the cells are filtered by a sterile 600-mesh screen and then sorted by an up-flow cytometer, and the cell population with green fluorescence is sorted into a pore plate; after the cell growth is recovered, establishing a library and storing the library as a BHK21-KO-IM cell population;
sorting the BHK21-KO-IM cell population into a 96-well plate by a flow cytometer for culture, wherein each well is provided with one cell clone; after the growth is recovered, adjusting the cell density of each hole to be approximately the same, and manufacturing a compound plate for comparing the virus proliferation efficiency after the cells are amplified;
removing culture supernatant, and respectively inoculating 200ul of virus proliferation maintenance liquid containing the same; observing cytopathic effect every day, completing virus proliferation in 72 hours, and measuring the titer of each virus proliferation sample after 3 times of freeze thawing; if the cell pathological changes exist, the cell pathological changes are judged to be positive, otherwise, the cell pathological changes are negative, the virus multiplication capacities of different cell clones are compared, and the cell clones with the improved multiplication capacity are obtained.
3. The method according to claim 1 or 2, further characterized in that the virus is selected from the group consisting of pseudorabies virus, japanese encephalitis virus, newcastle disease virus.
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