KR20160041148A - Immortalized porcine cell overexpressing telomerase reverse transcriptase and use thereof - Google Patents

Abstract

The present invention relates to an immortalized porcine cell line overexpressing telomerase reverse transcriptase and a method for producing the same. The present invention also relates to a method for screening gene scissors using the pig cell line.
The immortalized porcine cell line overexpressing the telomerase reverse transcriptase according to the present invention and the screening method of the gene scissors using the same or the method for confirming the performance of the gene scissors can be used for genetic research, It is expected that the pairing of gene scissors or gene scissors can be confirmed effectively, which can contribute greatly to the development of gene scissors.

Description

Immortalized porcine cell overexpressing telomerase reverse transcriptase and use thereof, wherein the telomerase reverse transcriptase is overexpressed,

The present invention relates to an immortalized porcine cell line and a method for producing the same.

The present invention also relates to a method for screening gene scissors using the pig cell line.

Because pigs are physiologically similar to human parts, they are valuable as biomedical model animals such as xenotransplantation. In addition, a somatic cell nuclear transfer technique using the transformed cells as a donor cell can be used to prepare a model animal necessary for such heterologous transplantation. To develop such a model animal, overexpression or knock-out of the target gene is required. To date, pigs overexpressing the desired gene have been knocked out using homologous recombination techniques, compared to only a few pigs (alpha-galactosidase, cystic fibrosis and IL2 receptor). The reason why knock-out models are difficult to produce is that the efficiency of homologous recombination is low. However, it is also an important obstacle that the cell itself has a limited lifetime to develop in vitro. To solve these two problems, there is a need for an efficient method of knock-out and immortalized cells.

Genetic scissors such as ZFN, TALEN and RGEN, which are currently being developed, make several pairs at the same time in order to make a sophisticated pair of gene scissors that cuts the target gene, and then directly acts on the cells to perform the most sophisticated and highly efficient gene scissors pair . However, other studies have reported that gene scissors that work well in cells of other species did not work in pigs. Therefore, the need for a pig cell line has been raised to make knock-out pigs of the target gene, and immortalized pig cells may be useful for selecting these pair of gene scissors.

Accordingly, the present inventors have made intensive efforts to invent a method for screening useful gene scissors or confirming the functions and effects of gene scissors. As a result, the present inventors have found that a pig cell line overexpressing a specific gene is effectively immortalized and this immortalized pig cell line The present inventors have completed the present invention based on the fact that screening of gene scissors can be effectively performed.

An aspect of the present invention is to provide an immortalized porcine cell line and a method for producing the same.

Another aspect of the present invention is to provide a method for screening gene scissors using the pig cell line.

One aspect of the invention provides an immortalized porcine cell line overexpressing telomerase reverse transcriptase (TERT).

Hereinafter, the present invention will be described in detail.

Telomere is a specific structure located at the end of a chromosome. It is a noncoding region with a repetitive nucleotide sequence of TTAGGG in vertebrates. It is a region from the fusion or deterioration with neighboring chromosomes, Lt; / RTI > Thus, the shortening of the chromosomal end, which is essential during chromosome replication, protects the gene located near the chromosome end from damage. As time passes, telomere ends become shorter and shorter due to cell division, eventually the genetic material at the end disappears and cells die. The shortening of the telomeric end is a phenomenon that occurs only in DNA replication of eukaryotes due to the problem of terminal replication. This loss of telomeres is supplemented by telomerase, in particular telomerase reverse transcriptase. The telomerase is a ribonucleoprotein, which is an enzyme that adds a repetitive DNA sequence to the 3 'end of a DNA strand at a telomere site found at the chromosome end of a eukaryotic cell, and its catalytic subunit is a telomerase reverse transcriptase (telomerase reverse transcriptase; TERT or hTERT for human).

The telomerase reverse transcriptase (TERT) of the present invention contains motifs found in various reverse transcriptases, and these motifs have been found to be conserved in a variety of species, from yeast to human (Meyerson et al. , Cell 90: 785-795,1997;

Nakamura et al., Science 277: 955-959,1997; Nugent and Lundblad, Genes & Dev. 12: 1073-1085, 1998). In addition, the C terminus of the telomerase reverse transcriptase contains a site in which CRM1 and 14-3-3 bind to human, and it has been found to be a necessary part in the formation of the fourth structure. The expression of human telomerase reverse transcriptase is found in about 85-95% of tumor tissues, which is a useful tool for diagnosing cancer. In addition, hTERT expression was found in the early stages of cancer in fatal cancers such as common cancers, thyroid cancer, breast cancer, cervical cancer, and prostate cancer.

The telomerase reverse transcriptase (TERT) may preferably be a human telomerase reverse transcriptase (hTERT).

The telomerase may be inserted into a recombinant vector and overexpressed in a porcine cell line.

The term " recombinant vector " as used herein refers to a gene product that contains an essential regulatory element operatively linked to express a desired gene or target RNA in a suitable host cell, so that the gene insert is expressed.

As used herein, the term " operably linked "refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA to perform a general function. Can affect the expression of a nucleic acid sequence that is operably linked to a promoter and a nucleic acid sequence that encodes a protein or RNA. The operative linkage with the recombination vector can be achieved using recombinant techniques well known in the art And site-specific DNA cleavage and linkage are performed using enzymes generally known in the art.

The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable expression vectors include signal sequence or leader sequences for membrane targeting or secretion, as well as expression control sequences such as promoter, operator, initiation codon, termination codon, polyadenylation signal and enhancer (promoter gene) . The promoter of the vector may be constitutive or inducible. The expression vector also includes a selection marker for selecting a host cell containing the vector and, if the expression vector is a replicable vector, a replication origin. The recombinant vector of the present invention may preferably be a pCMV-hTERT-IRES-DsRed vector.

The vector may be composed of the following cleavage map.

The term " overexpression " means a series of processes in which an exogenous gene, which is not found in a normal cell in a natural state, is introduced into an artificial manner for expression.

The term " immortalization " refers to a state in which a series of changes that may occur when a normal cell is cultured in vitro is changed. The change includes a change in the amount of time that the cell is doubled, a change in cell size, And a state in which an aspect has changed.

Introduction of a vector into a host cell can be accomplished by transfection, electroporation, transduction, microinjection, or ballistic introduction methods

The term " immortalization " refers to a state in which a series of changes that may occur when a normal cell is cultured in vitro is changed. The change includes a change in the amount of time that the cell is doubled, a change in cell size, And a state in which an aspect has changed.

The porcine cell line according to the present invention means a cell isolated from a pig, and the kind of the cell is not limited. For example, it may be a somatic cell, a germ cell, a progenitor cell, a tumor cell, or a stem cell, and may be a degenerated stem cell. Pigs are also not restricted, and may be adults, fetuses, carcasses, and the like. In the present invention, fibroblasts isolated from fetuses were used in the specific examples.

In addition, one aspect of the present invention provides a method for producing an immortalized porcine cell line comprising transforming a porcine cell line with a recombinant vector comprising a nucleotide encoding telomerase reverse transcriptase (TERT) .

The recombinant vector comprising the nucleotide encoding the telomerase reverse transcriptase (TERT) may be a recombinant vector comprising the following cleavage map.

In addition, the pig cell line may most preferably be a fetal fibroblast.

Another aspect of the present invention provides a method for screening gene scissors using the immortalized pig cell line or a method for checking the performance of gene scissors.

Specifically, a method for screening the gene scissors, or a method for confirming the performance of the gene scissors, may include treating any gene scissors or gene scissors pairs for a specific gene in an immortalized pig cell line.

In the present invention, " gene scissors " means engineered nuclease, which means a biological tool for cutting a double helix of a gene. In general, genetic scissors are enzymes designed to specifically cleave 18 to 40 nucleotides and cleave two strands of DNA. Using gene scissors, double strand breaks can be introduced into specific genes in plant and animal cells. In all cells there are two repair systems that effectively repair it: one is non-homologous end joining (NHEJ) and the other is homologous recombination (HR). Emergency copper bonding is an effective restoration system that keeps the cutting site intact, but often involves the insertion and removal of several base pairs (small insertion and deletion). Using this, mutations can be induced at a high rate of 1 to 90% in cells transfected with gene scissors. That is, it is possible to produce a cell line or a plant in which the function of the gene is knocked out by causing a frame-shift in a protein coding sequence of a specific gene. In addition, when two pairs of gene scissors are introduced into a cell at the same time and two DNA fragments are cut, DNA fragments of several thousands to millions of units are produced and structural changes such as redundancy, deletion, inversion, translocation, , Which allows you to edit and rearrange specific genomes as desired.

Homologous recombination is a sophisticated DNA repair system that does not involve indel, unlike the emergency copper junction. In general, the truncated double helix DNA repair is to recover the truncated portion by copying the genetic information using the DNA region having the same nucleotide sequence as the other chromosome (Chromatid) (Error-free DSB repair mechanism). Using this principle, a targeting vector containing a homologue arm similar to that of the truncated DNA can be introduced into the cell together with the gene scissors to easily induce or control the expression of a specific gene at a desired position, -in) cell lines or plants or plants.

Over the last two decades, genetic scissors have been used to identify and cut specific DNA sequences and to identify three types of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR / Cas-derived RNA guided endonuclease Respectively.

The screening method for genetic scissors or the method for confirming the performance of genetic scissors according to the present invention can effectively identify a pair of gene scissors or gene scissors that can be usefully used in genetic research, biotechnology, and gene therapy, It is expected that it will contribute greatly to the development of gene scissors.

The method may further include the step of determining whether a specific gene is knocked out, knocked in, or knocked down.

The screening method for genetic scissors or the method for confirming the performance of genetic scissors according to the present invention can effectively identify a pair of gene scissors or gene scissors that can be usefully used in genetic research, biotechnology, and gene therapy, It is expected that it will contribute greatly to the development of gene scissors.

Figure 1 A shows the vector map used to make the immortalized cells.
FIG. 1B is a graph comparing differences in times when the number of cells increases by two or more times as the number of control cells and immortalized cells increases.
FIG. 1C is a graph showing the difference in cell size with increasing number of passages.
Fig. 1D is a photograph comparing differences in shape of cells in each group.
E in Fig. 1 is a photograph showing a passage showing abnormality through karyotyping of cells according to the passage
FIG. 2A is a diagram showing that the TERT gene is properly inserted into the gene of the immortalized cell.
FIG. 2B is a view for confirming that the inserted gene is properly transcribed into mRNA.
FIG. 2C is a graph showing that the efficiency is significantly lowered as a result of somatic cell nuclear transfer using the cells thus constructed.
FIG. 2D is a graph showing real-time PCR results for identifying genes showing differences in expression patterns when immortalized cells are compared with control cells. FIG.
FIG. 3A is a diagram showing a position where a TALEN pair is to be joined and a position to be operated in a target gene.
FIG. 3B shows the results of T7E1 analysis for three colony knock-outs of various colonies.
FIG. 3C shows a sequence analysis result of three knock-outs.
Fig. 3D shows the fPCR results demonstrating that each colony is a definite biallelic knock-out.
FIG. 3E shows FACS results showing that the expression of the target gene disappeared in the three colony constructs.
Figure 4 is a table comparing the telomerase activity of control cells versus immortalized cells.
Fig. 5 is information on the primers used in the present experiment.
Figure 6 is information on the primers used in real-time PCR.
FIG. 7 is a photograph showing a process in which only one cell forms a colony.
FIG. 8 is a graph comparing the expression patterns of DNMT1, DNMT3a and DNMT3b, which are genes related to cell methylation, and GLUT1 and LDHA, metabolism related genes.
FIG. 9 is a sequencing result showing that the TERT gene is inserted in the immortalized cells.
FIG. 10 shows the result of T7E1 analysis in which knock-out was observed in 44 colonies out of a total of 116 colonies by progressing knock-out on the CMAH gene.
Figure 11 shows the results of biallelic knock-out at colony 13, 24 and 26 in red in the fPCR results for the CMAH knock-out colony.
FIG. 12 shows sequencing results of 282 bp inserted in the 24th colony in which CMAH Biallelic knock-out occurred.
FIG. 13 is a photograph of a blastocyst prepared using CMAH knock-out cells and the efficiency thereof.
FIG. 14A is a diagram showing a position at which a TALEN pair for joining a GGTA1 gene to knock-out and a position to be operative.
Figure 14B shows the results of T7E1 analysis for three colony knockout out of various colonies.
FIG. 14C shows a sequence analysis result of three knock-outs.
Figure 14D shows the fPCR results demonstrating that each colony is a biallelic knock-out.
Figure 15 shows the T7E1 analysis showing knock-out in 27 out of 57 colonies in the first GGTA1 knock-out experiment.
Figure 16 shows the fPCR results for the first knock-out experiment.
Figure 17 shows the T7E1 analysis showing that knock-out occurred in 26 out of 66 colonies in the second GGTA1 knock-out experiment.
Figure 18 shows the fPCR results for the second knock-out experiment.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Preparation and maintenance of control cells

  Mini - pig fetal fibroblast was used for control cell production. The recovered embryos were divided into three parts: the head, trunk and tail, and only the trunk part was used to make control cells.

The fetal body was washed three times with PBS and then finely ground in a 60 mm dish containing trypsin solution. The pulverized tissue was allowed to stand in an incubator at 37 ° C for 30 minutes and then centrifuged at 1,500 rpm for 2 minutes. The supernatant was discarded and the submerged tissue was washed with PBS and the same centrifugation procedure was repeated twice. (Gibco), 1% penicillin / streptomycin (P / S) (Gibco), 1% non-essential amino acid (NEAA) Dulbecco's Modified Eagle? Added with 100 mM? -Mercaptoethanol (ME)? Medium (DMEM). The medium containing the suspended tissue was allowed to stand at room temperature for 5 minutes. After 5 minutes, the supernatant was transferred to a cell culture dish, and the culture medium was replenished every 2-3 days, and the cells were completely grown until the culture dish was filled.

Example 2. Immortalization of control cells

The purified hTERT gene was inserted into the pCMV-IRES-DsRed vector for immortalization. The pCMV-IRES-DsRed vector with the hTERT gene inserted is shown in FIG. 1A. The inserted pCMV-hTERT-IRES-DsRed vector was added to the control cells using Fugene HD. After two days of transformation, 1000 μg / ml of neomycin (G418; Gibco) was treated for 7 days, and only surviving cells resistant to antibiotics were selected and subcultured (FIG. 1).

Example 3: Comparison of ability of control cells and immortalized cells

1) Time measurement in which cells are doubled

Control cells and immortalized cells were plated in a 12-well cell culture dish at 4 × 10 ⁴ cells / well. Cells in 4 wells are suspended with trypsin every 24 hours and the number of cells is measured using a hemacytometer. Then, the time for doubling the number of cells was measured by using a doubling time online calculator (http://www.doubling-time.com/compute.php) to a total of 24 passages for every three passages.

2) cell size

The cells in the hemacytometer are photographed under a 200x microscope. The size of 100 cells was measured by using ImageJ (http://rsbweb.nih.gov/ij/) program for a total of 24 passages in every three passages.

3) PCR

Genomic DNA was extracted using the G-spin Genomic DNA Extraction Kit (iNtRON Biotechnology, Gyeonggido, Korea). Amplification and confirmation of the inserted TERT gene was performed using Maxime PCR PreMix (i-StarTaq, iNtRON). Information on the primers and band sizes used is summarized in FIG.

4) Sequencing analysis

Sequencing of the target gene was carried out by inquiring to the relevant company (Macrogen Ltd., Seoul, Korea).

 5) Karyotype analysis

Analysis of the karyotypes of the target genes was carried out by inquiring to the relevant companies (GenDix, Inc., Seoul, Korea).

Example 4. Evaluation of ability of one cell to form colony

DMEM medium 20 supplemented with 15% FBS, 1% P / S, 1% NEAA and 100 mM? -ME containing immortalized cells in a lid of Falcon dish (Catalog number # 351006; Falcon, Franklin Lakes, New Jersey, USA) lt; RTI ID = 0.0 > ul < / RTI > To confirm that one cell is capable of forming a colony, one cell was transferred to a DMEM drop with 4 μl of 15% FBS, 1% P / S, 1% NEAA and 100 mM β-ME using a micromanipulator gave. After 7 days, the well-grown colonies were selected and cultured in 96-, 24-, and 6-well, 60-mm and 100-mm cell culture dishes, respectively.

Example 5: Evaluation of gene expression

Total RNA was extracted using easy-spin Total RNA Extraction Kit (iNtRON). And complementary DNAs (cDNAs) were synthesized using Maxime RT Premix (iNtRON). Information on these primers was generated in Fig. The gene expression pattern was confirmed by p53, p16, Bax and Bcl-xl using real time PCR machine (7300 Real-Time PCR System; Applied Biosystems).

Example 6. Knock-out of CMAH gene using TALEN and isolation of the cells

1 × 10 ⁶ immortalized cells were transformed with 30 μl of Turbofect ^™ (Fermentas Inc., Maryland, USA) and 10 μg of the gene scissors vector. Transformed cells were cultured for 2 days and then electroporated.

Example 6. T7E1 assay

Genomic DNA was extracted three days after transformation using the G-DEX IIc Genomic DNA Extraction Kit (iNtRON). The portion where the gene scissors acted was amplified using the primers listed in FIG. T7E1 analysis proceeded as described in the previous paper.

Example 7. Fluorescent PCR (fPCR) analysis

Carboxyfluorescein (FAM) was customized with the 5 'portion of the forward primer attached (Bioneer Corporation, Daejon, South Korea). The amplified PCR products were fragment-separated by capillary electrophoresis using ABI 3730xl with POP-7 polymer. GeneScan Rox500 size standard (Life technologies, NY, US) was used as an internal size marker. Analytical samples were denatured at 95 ° C for 5 minutes and analyzed using a Genetic Analyzer. The results were analyzed by peak height analysis using pick scanner software v1.0 (Life technologies).

Example 8. FACS analysis

CMAH biallelic knock-out cells were floated on PBS supplemented with 0.1% BSA, and then the final concentration was adjusted to 5 × 10 ⁵ to 1 × 10 ⁶ cells / ml. The mixture was mixed with Anti-Neu5Gc (Sialix, Massachusetts, USA) and allowed to stand for 20 minutes on ice. After the incubation, the cells were washed with PBS supplemented with 0.1% BSA twice, and the prepared cells were subjected to FACS.

FIG. 1B compares the difference in time when the number of cells increases by two times as the passage of control cells and immortalized cells increases. As shown in Fig. 1B, the doubling time of the number of control cells was 46.4 ± 1.1 hours, whereas that of immortalized cells was doubled in 26.9 ± 0.6 hours.

FIG. 1C is a graph comparing cell sizes of control cells versus immortalized cells with increasing number of passages. As shown in FIG. 1C, the size of immortalized cells was always kept below 20 μm (average 17.9 ± 0.2) regardless of the increase in the number of passages. However, in the case of control cells, the size of the cells exceeded 20 μM over 12 passages and began to show a significant difference when compared to the same passage of immortalized cells. In contrast to immortalized cells, the size of control cells was continuously increased over 12 passages.

Fig. 1D compares differences in cell shape between control cells and immortalized cells. As shown in Fig. 1D, it was confirmed that the shape of the control cells gradually increased in comparison with the initial three passages. On the other hand, immortalized cells did not increase in cell size, but they were observed to change the pattern of cell growth as a specific cluster.

FIG. 1E compares the results of cell karyotype analysis of control cells versus immortalized cells. As shown in FIG. 1E, in the immortalized cells, abnormal karyotypes could be identified as the passage progressed.

 FIG. 2A is an A diagram showing that the TERT gene is properly inserted into the gene of the immortalized cell.

FIG. 2B shows that the inserted gene is properly transcribed into mRNA.

FIG. 2C is a graph showing that efficiency is significantly lowered as a result of somatic cell nuclear transfer using recombinant cells.

FIG. 2D is a D graph showing a real-time PCR result for identifying a gene showing a difference in expression pattern when immortalized cells are compared with control cells. As shown in Fig. 2D, it was confirmed that, in the case of p53 expression, the expression was kept constant regardless of the increase in the number of lines as a whole. This pattern was confirmed in Bcl-xl as well. However, in the case of p16 and Bax, the expression in immortalized cells tended to decrease as compared with the control cells as the number of passages increased. It was confirmed that the immortalized cells immortalized by controlling the gene associated with p16, not p53, were immortalized without stopping the cell cycle, and the apoptosis was inhibited based on the decrease of the expression of proapoptotic gene Bax in immortalized cells .

 FIG. 3A is a diagram showing a position where a TALEN pair is to be joined and a position to be operated in a target gene.

Figure 3B shows the T7E1 assay for three colony knock-outs out of various colonies.

FIG. 3C shows the result of sequence analysis for three knock-out colonies.

D in Fig. 3 is the fPCR result demonstrating that each colony is a definite biallelic knock-out.

FIG. 3E shows FACS results showing that the expression of the target gene disappeared in the three colony constructs.

 Figure 4 is a table comparing the telomerase activity of control cells versus immortalized cells. As can be seen from Fig. 4, the activity of telomerase in immortalized cells was significantly higher than that of the control.

FIG. 7 is a photograph showing a process in which a single cell of an immortal cell line actually forms a colony.

 FIG. 8 is a graph comparing the expression patterns of DNMT1, DNMT3a and DNMT3b, which are genes related to cell methylation, and GLUT1 and LDHA, metabolism related genes. As shown in Fig. 8, no change in the gene related to the methylation of the cell could be found even when the number of the cells increased. However, it can be observed that GLUT1 is increased, which is expected to result in the immortalized cells uptake a larger amount of glucose than the control cells. On the other hand, the increase in LDHAs, which may occur as a result of apoptosis or necrosis, in the control group also suggests that more cell death occurred in control cells than in immortalized cells.

 FIG. 9 is a sequencing result showing that the TERT gene is inserted in the immortalized cells.

 FIG. 10 shows the result of T7E1 analysis in which knock-out was observed in 44 colonies out of a total of 116 colonies by progressing knock-out on the CMAH gene.

 Figure 11 shows the results of biallelic knock-out at colony 13, 24 and 26 in red in the fPCR results for the CMAH knock-out colony.

 FIG. 12 shows sequencing results of 282 bp inserted in the 24th colony in which CMAH Biallelic knock-out occurred.

FIG. 13A is a photograph of a blastocyst prepared using CMAH knock-out cells.

FIG. 13B is a view comparing the control cell line with the immortalized cell line with respect to the blastocyst expression efficiency.

 FIG. 14A is a diagram showing a position at which a TALEN pair for joining a GGTA1 gene to knock-out and a position to be operative.

FIG. 14B shows the results of T7E1 analysis for three colony knock-outs out of a plurality of colonies.

FIG. 14C shows a sequence analysis result of three knock-outs.

Fig. 14D shows fPCR results demonstrating that each colony is a biallelic knock-out.

 Figure 15 shows the T7E1 analysis showing knock-out in 27 out of 57 colonies in the first GGTA1 knock-out experiment.

 Figure 16 shows the fPCR results for the first knock-out experiment.

 Figure 17 shows the T7E1 analysis showing that knock-out occurred in 26 out of 66 colonies in the second GGTA1 knock-out experiment.

 Figure 18 shows the fPCR results for the second knock-out experiment.

Claims (12)

Immortalized porcine cell line overexpressing telomerase reverse transcriptase (TERT). The porcine cell line according to claim 1, wherein the telomerase reverse transcriptase is human. The swine cell line according to claim 1, wherein the swine cell line is transformed by a recombinant vector comprising the following cleavage map.

The swine cell line according to claim 1, wherein the swine cell line is a swine fibroblast. The swine cell line according to claim 1, wherein the swine cell line is derived from a fetus of a pig. The swine cell line according to claim 1, wherein the undifferentiated swine cell line has increased expression of p16 and Bax compared to a normal cell line. A method for producing an immortalized porcine cell line comprising transforming a porcine cell line with a recombinant vector comprising a nucleotide encoding telomerase reverse transcriptase (TERT). [Claim 7] The method according to claim 7, wherein the recombinant vector is a recombinant vector comprising the following cleavage map.

[Claim 7] The method according to claim 7, wherein the porcine cell line is a fetal fibroblast. A method for screening gene scissors, comprising treating a porcine cell line of any one of claims 1 to 6 with any gene scissors or gene scissors pair for a specific gene. The method of claim 10,
Determining whether a specific gene is knocked out, knocked in, or knocked down. A method for identifying the performance of a gene scissor comprising the step of treating any of the gene scissors or gene scissors pairs for a particular gene in the porcine cell line of any one of claims 1 to 6.

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