238 PRODUCTION OF CLEAVAGE-RESISTANT PHYTASE TRANSGENIC PIGS BY HANDMADE CLONING

2016 ◽  
Vol 28 (2) ◽  
pp. 251
Author(s):  
M. Zhang ◽  
S. Chen ◽  
X. Chen ◽  
Y. Huang ◽  
L. Wei ◽  
...  
Keyword(s):  
Cell Lines ◽  
Animal Husbandry ◽  
Phytase Activity ◽  
Specific Gene ◽  
Transgenic Pigs ◽  
Specific Expression ◽  
Phytase Gene ◽  
Tissue Specific ◽  
Fetal Fibroblasts ◽  
Handmade Cloning

Rapidly developing and intensive animal husbandry of livestock is a major contributor to global environmental pollutions. Large quantities and high concentrations of manure waste that contains phytate phosphorus are generated. The use of phytase can effectively solve the problem of high phosphorus pollution in the fecal material of monogastric animals. Enviropigs, producing phytase in the salivary glands and secreting the enzyme in the saliva, were first generated at the University of Guelph (Guelph, ON, Canada) in 1999. However, phytase is easily inactivated in digestive processing. To address this problem, we improved the transgene construct and successfully generated phytase transgenic pigs by handmade cloning. The Escherichia coli periplasmic phosphoanhydride phosphohydrolase (appA) gene was subcloned. Using fragment substitution method, we designed a phytase gene that was insensitive to cleavage by pepsin and trypsin and had a higher affinity for the substrate. After codon optimization, the designed phytase gene was named Cafp and subcloned downstream of the pig parotid secretory protein (PSP) gene promoter. The tissue-specific vector p-PSP-Intron-Cafp was constructed and transferred into Landrace fetal fibroblasts by electroporation. The cell lines carrying Cafp were used as nuclear donors in handmade cloning. Cloned embryos were cultured in vitro to blastocysts and transferred to recipient sows. The presence of Cafp was tested by PCR and sequencing of cloned pigs. Phytase activity in saliva, feed, and feces was detected by the ammonium molybdate method with a slight modification. Immunohistochemistry (IHC) was used to determine tissue-specific expression. Three cell lines carrying Cafp were obtained. We generated 1027 blastocysts; 712 were of good quality and transferred to 6 recipients. Fourteen piglets were born, of which 6 survived. The PCR and sequencing results showed that 7 (3 live and 4 dead) of the 14 piglets carried Cafp. Phytase activity in the saliva of the 6 live cloned pigs was tested at 4 months of age and only 1 pig had 0.155 FTU mL–1 enzyme activity. The enzyme in the other 2 pigs may be inactivated in the transgenic parotid gland. Among all the transgenic pigs, the highest phosphorus digestion rate was 59.2% of intake, which represents a 25.4% decrease in fecal emissions compared with the average of controls. The IHC results on the 3 later dead, Cafp-positive pigs showed that the transgene was expressed only in parotids, confirming tissue-specific gene expression. In summary, cleavage-resistant phytase transgenic pigs were successfully produced through handmade cloning. The cloned pigs offer a unique biological approach to manage phosphorus nutrition and environmental pollution in animal husbandry.

scholarly journals Methylation of the Cyclin A1 Promoter Correlates with Gene Silencing in Somatic Cell Lines, while Tissue-Specific Expression of Cyclin A1 Is Methylation Independent

2000 ◽  
Vol 20 (9) ◽  
pp. 3316-3329 ◽  
Author(s):  
Carsten Müller ◽  
Carol Readhead ◽  
Sven Diederichs ◽  
Gregory Idos ◽  
Rong Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Promoter Methylation ◽  
Cpg Island ◽  
Trichostatin A ◽  
Specific Gene ◽  
Cyclin A1 ◽  
Specific Expression ◽  
Tissue Specific ◽  
Tissue Specific Expression

ABSTRACT Gene expression in mammalian organisms is regulated at multiple levels, including DNA accessibility for transcription factors and chromatin structure. Methylation of CpG dinucleotides is thought to be involved in imprinting and in the pathogenesis of cancer. However, the relevance of methylation for directing tissue-specific gene expression is highly controversial. The cyclin A1 gene is expressed in very few tissues, with high levels restricted to spermatogenesis and leukemic blasts. Here, we show that methylation of the CpG island of the human cyclin A1 promoter was correlated with nonexpression in cell lines, and the methyl-CpG binding protein MeCP2 suppressed transcription from the methylated cyclin A1 promoter. Repression could be relieved by trichostatin A. Silencing of a cyclin A1 promoter-enhanced green fluorescent protein (EGFP) transgene in stable transfected MG63 osteosarcoma cells was also closely associated with de novo promoter methylation. Cyclin A1 could be strongly induced in nonexpressing cell lines by trichostatin A but not by 5-aza-cytidine. The cyclin A1 promoter-EGFP construct directed tissue-specific expression in male germ cells of transgenic mice. Expression in the testes of these mice was independent of promoter methylation, and even strong promoter methylation did not suppress promoter activity. MeCP2 expression was notably absent in EGFP-expressing cells. Transcription from the transgenic cyclin A1 promoter was repressed in most organs outside the testis, even when the promoter was not methylated. These data show the association of methylation with silencing of the cyclin A1 gene in cancer cell lines. However, appropriate tissue-specific repression of the cyclin A1 promoter occurs independently of CpG methylation.

scholarly journals Isolation of a novel macrophage-specific gene by differential cDNA analysis

Blood ◽  
1995 ◽  
Vol 85 (6) ◽  
pp. 1620-1629 ◽  
Author(s):  
K Spilsbury ◽  
MA O'Mara ◽  
WM Wu ◽  
PB Rowe ◽  
G Symonds ◽  
...  
Keyword(s):  
Cell Lines ◽  
Core Protein ◽  
Signal Sequence ◽  
Fetal Liver ◽  
Computer Assisted ◽  
Specific Gene ◽  
Local Similarity ◽  
Specific Expression ◽  
Reading Frame ◽  
Cdna Analysis

To analyze myelomonocytic differentiation we have used the approach of differential cDNA analysis to isolate novel genes that are preferentially expressed in mature macrophages. Differential screening of a macrophage cDNA library led to the identification of a novel cDNA that showed macrophage lineage- and differentiation stage-specific expression. Transcripts from the gene, which we have termed Mpg-1, are found at a high level in mature human and murine macrophages and at a moderate level in certain myelomonocytic cell lines. The expression of Mpg-1 was found to increase when murine fetal liver hematopoietic progenitor cells were induced to differentiate into macrophages. An Mpg- 1-specific transcript was not detected in a wide variety of other tissues and cell lines. The DNA sequence of Mpg-1 (4,214 bp) was obtained from a series of overlapping cDNA, 33′ rapid amplification of cDNA ends (RACE), and genomic clones. Primer extension analysis predicted the existence of multiple transcription start sites, ranging from 26 to 117 bp upstream of the 53′ proximal ATG of the open reading frame. The predicted 669-amino acid, Mpg-1-encoded protein has potential glycosylation and phosphorylation sites in addition to a signal sequence. The core protein is predicted to have a molecular weight of 71 to 74 kD. Computer-assisted local similarity searches indicate that Mpg-1 is a novel gene that may share a distant ancestry to perforin, a lytic protein found in cytotoxic T lymphocytes and natural killer cells.

MULTI-TRANSGENIC PIGS FOR XENOTRANSPLANTATION

2013 ◽  
Vol 25 (1) ◽  
pp. 320 ◽  
Author(s):  
David Ayares
Keyword(s):  
Cellular Response ◽  
Nonhuman Primates ◽  
Genetic Manipulation ◽  
Humoral Response ◽  
Specific Gene ◽  
Transgenic Pigs ◽  
Tissue Specific ◽  
Xenograft Rejection ◽  
Galactosyl Transferase

Successful development of somatic cell nuclear transfer (cloning) technology in pigs has allowed for precise genetic manipulation of the pig genome. For xenotransplantation applications, pigs have been produced in which both copies of the α1,3-galactosyl transferase (GT) gene were inactivated (GTKO pigs). Analysis of tissues from GTKO pigs demonstrated a complete lack of immunogenic Galα1,3Gal (Gal) sugars, while in vivo pre-clinical studies in nonhuman primates, using cells (i.e. pancreatic islets) or whole organs (heart, kidney, liver, lung), demonstrated the elimination of hyperacute rejection, and prolonged survival compared to wild-type controls. While survival of GTKO xenografts was extended, challenges including induced antibody responses to non-gal antigens, thrombosis, inflammation, and cell-mediated rejection remained, pointing to the need for further genetic modification of the source pig. Towards this goal, through a combination of cloning and breeding, in combination with GTKO, we have produced multi-transgenic pigs (some with 5 different transgenes) with controlled expression of genes for (1) complement regulation to address the humoral response to anti-non-gal targets (DAF, CD46); (2) inhibition of inflammation and thrombosis (TFPI, CD39, thrombomodulin, EPCR); and (3) local protection against the human cellular response (CTLA4Ig, CIITA-DN). For some transgenes a constitutive promoter system can be used for expression in all tissues, such that one animal can be used for multiple transplant applications, however, our results have shown that for certain transgenes, tissue-specific gene expression is preferred. Since inhibition of thrombosis, complement modulation, and suppression of T-cell responses are important to delayed xenograft rejection of both whole organs and islet cell xenografts, pigs have been produced with tissue-specific transgene expression in either the vascular endothelium or endocrine pancreas compartments, or constitutively in all tissues. In vivo results in nonhuman primates have demonstrated complete normalization of blood glucose for up to 1 year in diabetic monkeys, and 8-month survival of multigenic pig hearts in baboons, evidence for the promise of this technology for human clinical applications.

Regulation of lineage-specific transcription of the sucrase-isomaltase gene in transgenic mice and cell lines

1995 ◽  
Vol 269 (6) ◽  
pp. G925-G939 ◽  
Author(s):  
A. J. Markowitz ◽  
G. D. Wu ◽  
A. Bader ◽  
Z. Cui ◽  
L. Chen ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cell Lines ◽  
Reporter Gene ◽  
Molecular Mechanisms ◽  
Ectopic Expression ◽  
Transgenic Animals ◽  
Intestinal Cell ◽  
Specific Gene ◽  
Specific Expression ◽  
Endogenous Gene

Sucrase-isomaltase (SI), a gene expressed exclusively in absorptive enterocytes, was used to examine the molecular mechanisms that regulate cell-specific gene expression in the intestinal epithelium. Transgenic mice were made with a construct containing nucleotides -8,500 to +54 of the mouse SI gene linked to a human growth hormone reporter gene. In adult transgenic animals, high-level transgene expression was limited to the small intestine, with low levels of ectopic expression in the colon. In contrast to the endogenous gene that is expressed only in enterocytes, the transgene was expressed in all four cell lineages, including enterocytes, enteroendocrine, goblet, and Paneth cells. To examine this process of lineage-specific expression further we studied Caco-2 and COLO DM cell lines, which model enterocytes and enteroendocrine cells, respectively. Reminiscent of results in transgenic animals, only Caco-2 cells transcribed the endogenous SI gene, whereas both Caco-2 and COLO DM cells supported transcription from chimeric SI reporter gene constructs. Taken together, these data suggest that each intestinal cell lineage has the cellular machinery to transcribe the SI gene. Moreover, these findings imply that transcription is normally repressed in nonenterocytic cells, possibly via a transcriptional silencer residing outside of the region of the SI gene examined in these studies.

scholarly journals Transcriptomic Analysis and Specific Expression of Transcription Factor Genes in the Root and Sporophyll of Dryopteris fragrans (L.) Schott

2020 ◽  
Vol 21 (19) ◽  
pp. 7296
Author(s):  
Lingling Chen ◽  
Dongrui Zhang ◽  
Chunhua Song ◽  
Hemeng Wang ◽  
Xun Tang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Glandular Trichomes ◽  
Transcriptomic Analysis ◽  
Specific Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Transcription Factor Genes ◽  
Dryopteris Fragrans ◽  
Different Tissues

Background: Dryopteris fragrans, which is densely covered with glandular trichomes, is considered to be one of the ferns with the most medicinal potential. The transcriptomes from selected tissues of D. fragrans were collected and analyzed for functional and comparative genomic studies. The aim of this study was to determine the transcriptomic characteristics of wild D. fragrans sporangium in tissues from the SR (root), SL (sporophyll), and TRL (sporophyll with glandular trichomes removed). Results: Cluster analysis identified genes that were highly expressed in an organ-specific manner according to read mapping, feature counting, and normalization. The functional map identified gene clusters that can uniquely describe the function of each tissue. We identified a group of three tissue-specific transcription factors targeting the SL, SR, and TRL. In addition, highly expressed transcription factors (TFs) were found in each tissue-specific gene cluster, where ERF and bHLH transcription factors were the two types showing the most distinct expression patterns between the three different tissues. The specific expression of transcription factor genes varied between the different types of tissues. The numbers of transcription factors specifically expressed in the roots and sporophylls were 60 and 30, respectively, while only seven were found for the sporophylls with glandular trichomes removed. The expression of genes known to be associated with the development of glandular trichomes in flowering plants, including MIXTA, ATML1, and MYB106, were also validated and are discussed. In particular, a unigene encoding MIXTA was identified and exhibited the highest expression level in SL in D. fragrans. Conclusions: This study is the first report of global transcriptomic analysis in different tissues of D. fragrans, and the first to discuss these findings in the context of the development of homologous glandular trichomes. These results set the stage for further research on the development, stress resistance, and secondary metabolism of D. fragrans glandular trichomes.

Differential DNA Methylation of TUBB1 Correlates with Tissue-Specific Hβ-1 Tubulin Expression

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4796-4796
Author(s):  
Paul Andrew Basciano ◽  
Rita Shaknovich ◽  
Maria E Figueroa ◽  
Paraskevi Giannakakou
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Promoter Region ◽  
Specific Expression ◽  
Tissue Specific ◽  
Methylation Difference ◽  
Intron 1 ◽  
Upstream Promoter Region ◽  
Exon 4 ◽  
Epigenetic Modulation

Abstract Abstract 4796 Introduction: Hβ-1 tubulin is essential for normal thrombopoiesis and constitutes the majority of β-tubulin within the platelet marginal band. Hβ-1 tubulin has the highest sequence diversity from other tubulin isotypes, and has expression restricted to hematologic tissues. The best-characterized expression occurs in megakaryocytes, where temporal expression is limited to specific stages of megakaryocyte maturation. While expression of Hβ-1 tubulin has been shown to be dependent on transcription factors such as NF-E2, these factors have a multitude of gene targets and do not provide a full explanation of mechanisms responsible for specific Hβ-1 tubulin expression. Epigenetic regulation of gene transcription is now widely accepted as a mechanism of tissue-specific expression of gene products; however, the role of epigenetic regulation in the expression of tubulin isotypes has not been explored. Given the highly regulated expression of Hβ-1 tubulin and the potential of epigenetic modifications to drive specific gene expression, we hypothesized that epigenetic modulation via DNA methylation may be a mechanism for regulation of Hβ-1 tubulin expression. Methods: We identified 3 CpG-dense areas upstream and within TUBB1 (which encodes Hβ-1 tubulin) based on Human hg19 genome assembly. These areas included the putative promoter region (-2000 to +1bp from the 5'UTR) upstream of the transcriptional start site; a 3kb region spanning most of intron 1; and a region within exon 4. We used Sequenom MassARRAY EpiTYPER on bisulphite-converted DNA to quantitatively determine percent methylation at each CpG within the three CpG-dense regions. Specifically, we extracted genomic DNA from three cell lines expressing Hβ-1 tubulin (K562, MEG-01, and HEL), and six non-expressing cell lines (two hematologic cell lines (REH, KCL-22) and four epithelial cell lines (H1299, PC3, LNCap2, MDA-MB-231)), and performed bisulphite conversion of DNA. A difference of >25% was considered significant when comparing individual CpGs; methylation differences between CpGs across cell lines are reported as mean ± SEM. For each region, median methylation for each CpG was calculated across cell lines within a group, and regional methylation differences compared using the Mann-Whitney test. Results: Between 50–75% of CpGs within the three CpG-dense regions were available for analysis. In exon 4, there was no difference in overall methylation or methylation at any individual CpG between the Hβ-1 tubulin-expressing and non-expressing cell lines. In contrast, within intron 1, Hβ-1 tubulin-expressing cell lines were significantly hypomethylated compared to non-expressing cell lines (p=0.002). This difference was localized to a 1.5kb region within intron 1; mean methylation difference at each CpG within this region was 59±6%. The upstream promoter region similarly showed significant hypomethylation in Hβ-1 tubulin-expressing cell lines (p=0.001); the differentially methylated CpGs were localized to a 350bp region just upstream of the transcription start site, and mean methylation difference was 60±16% at each CpG. Methylation patters were highly similar between CpGs within each group (i.e. Hβ-1 tubulin-expressing or non-expressing cell lines), with only 10% of individual CpGs showing >15% methylation difference between cell lines of the same group. Conclusion: We found significant extra- and intra-genic DNA methylation differences in TUBB1 between Hβ-1 tubulin-expressing and non-expressing cell lines. Methylation changes were localized to two CpG-dense regions, namely the upstream promoter region and intron 1 of TUBB1, while a third region in exon 4 showed no differences in methylation. The overall methylation differences within the regions were attributable to large methylation differences at individual CpGs localized to particular areas within those regions. Taken together, these results suggest that tissue-specific expression of Hβ-1 tubulin may be regulated in part by highly-specific changes in DNA methylation of TUBB1. To our knowledge, this is the first report of epigenetic modulation associated with tissue-specific tubulin isotype expression. Further work is underway to confirm these findings in normal primary hematopoietic tissues and to investigate their associations with temporal expression of Hβ-1 tubulin during megakaryocyte development. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Placenta-specific Expression of the Interleukin-2 (IL-2) Receptor β Subunit from an Endogenous Retroviral Promoter

2011 ◽  
Vol 286 (41) ◽  
pp. 35543-35552 ◽  
Author(s):  
Carla J. Cohen ◽  
Rita Rebollo ◽  
Sonja Babovic ◽  
Elizabeth L. Dai ◽  
Wendy P. Robinson ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Interleukin 2 ◽  
Endogenous Retroviruses ◽  
Specific Gene ◽  
Β Subunit ◽  
Gene Encoding ◽  
Specific Expression ◽  
Tissue Specific ◽  
Cellular Genes ◽  
Villous Trophoblast

The long terminal repeat (LTR) sequences of endogenous retroviruses and retroelements contain promoter elements and are known to form chimeric transcripts with nearby cellular genes. Here we show that an LTR of the THE1D retroelement family has been domesticated as an alternative promoter of human IL2RB, the gene encoding the β subunit of the IL-2 receptor. The LTR promoter confers expression specifically in the placental trophoblast as opposed to its native transcription in the hematopoietic system. Rather than sequence-specific determinants, DNA methylation was found to regulate transcription initiation and splicing efficiency in a tissue-specific manner. Furthermore, we detected the cytoplasmic signaling domain of the IL-2Rβ protein in the placenta, suggesting that IL-2Rβ undergoes preferential proteolytic cleavage in this tissue. These findings implicate novel functions for this cytokine receptor subunit in the villous trophoblast and reveal an intriguing example of ancient LTR exaptation to drive tissue-specific gene expression.

scholarly journals Starvation resistance and tissue-specific gene expression of stress-related genes in a naturally inbred ant population

2016 ◽  
Vol 3 (4) ◽  
pp. 160062 ◽  
Author(s):  
Nick Bos ◽  
Unni Pulliainen ◽  
Liselotte Sundström ◽  
Dalial Freitak
Keyword(s):  
Gene Expression ◽  
Stress Responses ◽  
Specific Gene ◽  
Starvation Resistance ◽  
Specific Expression ◽  
Tissue Specific ◽  
Trade Offs ◽  
Stress Related Genes ◽  
The Individual ◽  
Different Tissues

Starvation is one of the most common and severe stressors in nature. Not only does it lead to death if not alleviated, it also forces the starved individual to allocate resources only to the most essential processes. This creates energetic trade-offs which can lead to many secondary challenges for the individual. These energetic trade-offs could be exacerbated in inbred individuals, which have been suggested to have a less efficient metabolism. Here, we studied the effect of inbreeding on starvation resistance in a natural population of Formica exsecta ants, with a focus on survival and tissue-specific expression of stress, metabolism and immunity-related genes. Starvation led to large tissue-specific changes in gene expression, but inbreeding had little effect on most of the genes studied. Our results illustrate the importance of studying stress responses in different tissues instead of entire organisms.

scholarly journals A non-coding genetic variant maximally associated with serum urate levels is functionally linked to HNF4A-dependent PDZK1 expression

2018 ◽  
Author(s):  
Sarada Ketharnathan ◽  
Megan Leask ◽  
James Boocock ◽  
Amanda J. Phipps-Green ◽  
Jisha Antony ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepg2 Cells ◽  
Molecular Mechanisms ◽  
Genetic Variant ◽  
Fluorescent Protein ◽  
Serum Urate ◽  
Specific Gene ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Tissue Specific

ABSTRACTSeveral dozen genetic variants associate with serum urate levels, but the precise molecular mechanisms by which they affect serum urate are unknown. Here we tested for functional linkage of the maximally-associated genetic variant rs1967017 at the PDZK1 locus to elevated PDZK1 expression.We performed expression quantitative trait locus (eQTL) and likelihood analyses followed by gene expression assays. Zebrafish were used to determine the ability of rs1967017 to direct tissue-specific gene expression. Luciferase assays in HEK293 and HepG2 cells measured the effect of rs1967017 on transcription amplitude.PAINTOR analysis revealed rs1967017 as most likely to be causal and rs1967017 was an eQTL for PDZK1 in the intestine. The region harboring rs1967017 was capable of directly driving green fluorescent protein expression in the kidney, liver and intestine of zebrafish embryos, consistent with a conserved ability to confer tissue-specific expression. The urate-increasing T-allele of rs1967017 strengthens a binding site for the transcription factor HNF4A. siRNA depletion of HNF4A reduced endogenous PDZK1 expression in HepG2 cells. Luciferase assays showed that the T-allele of rs1967017 gains enhancer activity relative to the urate-decreasing C-allele, with T-allele enhancer activity abrogated by HNF4A depletion. HNF4A physically binds the rs1967017 region, suggesting direct transcriptional regulation of PDZK1 by HNF4A.With other reports our data predict that the urate-raising T-allele of rs1967017 enhances HNF4A binding to the PDZK1 promoter, thereby increasing PDZK1 expression. As PDZK1 is a scaffold protein for many ion channel transporters, increased expression can be predicted to increase activity of urate transporters and alter excretion of urate.

scholarly journals A Floxed exon (Flexon) approach to Cre-mediated conditional gene expression

2021 ◽  
Author(s):  
Justin M Shaffer ◽  
Iva Greenwald
Keyword(s):  
Gene Expression ◽  
Specific Protein ◽  
Specific Gene ◽  
Coding Region ◽  
Specific Expression ◽  
Control Of Gene Expression ◽  
Tissue Specific ◽  
Nonsense Mediated Decay ◽  
Conditional Gene Expression ◽  
Stop Cassette

Conditional gene expression allows for genes to be manipulated and lineages to be marked during development. In the established "lox-stop-lox" approach, Cre-mediated tissue-specific gene expression is achieved by excising the stop cassette, a lox-flanked translational stop that is inserted into the 5' untranslated region of a gene to halt its expression. Although lox-stop-lox has been successfully used in many experimental systems, the design of traditional stop cassettes also has common issues and limitations. Here, we describe the Floxed exon (Flexon), a stop cassette within an artificial exon that can be inserted flexibly into the coding region of any gene to cause premature termination of translation and nonsense-mediated decay of the mRNA. We demonstrate its efficacy in C. elegans by showing that, when promoters that cause weak and/or transient cell-specific expression are used to drive Cre in combination with a gfp(flexon) transgene, strong and sustained expression is obtained in specific lineages. We also describe several potential additional applications for using Flexon for developmental studies, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation or RNAi, and generation of genetic mosaics. The Flexon approach should be feasible in any system where any site-specific recombination-based method may be applied.

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