Synthetic sequence entanglement augments stability and containment of genetic information in cells

Science ◽  
2019 ◽  
Vol 365 (6453) ◽  
pp. 595-598 ◽  
Author(s):  
Tomasz Blazejewski ◽  
Hsing-I Ho ◽  
Harris H. Wang
Keyword(s):  
Genetic Information ◽  
Recombinant Dna ◽  
Fitness Landscape ◽  
Essential Gene ◽  
Computational Design ◽  
Genetically Engineered ◽  
Toxin Gene ◽  
Overlapping Genes ◽  
Synonymous Mutations ◽  
Natural Mutation

In synthetic biology, methods for stabilizing genetically engineered functions and confining recombinant DNA to intended hosts are necessary to cope with natural mutation accumulation and pervasive lateral gene flow. We present a generalizable strategy to preserve and constrain genetic information through the computational design of overlapping genes. Overlapping a sequence with an essential gene altered its fitness landscape and produced a constrained evolutionary path, even for synonymous mutations. Embedding a toxin gene in a gene of interest restricted its horizontal propagation. We further demonstrated a multiplex and scalable approach to build and test >7500 overlapping sequence designs, yielding functional yet highly divergent variants from natural homologs. This work enables deeper exploration of natural and engineered overlapping genes and facilitates enhanced genetic stability and biocontainment in emerging applications.

EMBRYO MANIPULATION AND GENE TRANSFER IN LIVESTOCK

1985 ◽  
Vol 65 (3) ◽  
pp. 527-538 ◽  
Author(s):  
R. B. CHURCH ◽  
F. J. SCHAUFELE ◽  
K. MECKLING
Keyword(s):  
Gene Transfer ◽  
Genetic Engineering ◽  
Embryo Transfer ◽  
Dna Sequences ◽  
Recombinant Dna ◽  
Fusion Gene ◽  
Bovine Genome ◽  
Monozygotic Twins ◽  
Genetically Engineered ◽  
Livestock Species

In the past few years significant progress has been made in manipulation of reproduction and in development of genetic engineering techniques which can be applied to animal species. Artificial insemination and embryo transfer are now used widely in the livestock industry. The advent of non-surgical embryo collection and transfer, embryo freezing and splitting along with estrus synchronization has allowed the industry to move from the laboratory to the farm. Embryo manipulation now involves embryo splitting to produce monozygotic twins, in vitro fertilization, cross-species fertilization, embryo sexing, and chimeric production of tetraparental animals among others. Advances in recombinant DNA, plasmid construction and embryo manipulation technologies allow the production of genetically engineered animals. The application of recombinant DNA technology involves the isolation and manipulation of desired genes which have potential for significant changes in productivity in genetically engineered livestock. Recombinant DNA constructs involve the coupling of promoter, enhancer, regulatory and structural DNA sequences to form a "fusion gene" which can then be multiplied, purified, assayed and expressed in cell culture prior to being introduced into an animal genome. Such DNA gene constructs are readily available for many human and mouse genes. However, they are not readily available for livestock species because the detailed molecular biology has not yet been established in these species. Gene transfer offers a powerful new tool in animal research. Transfer of genes into the bovine genome has been accomplished. However, successful directed expression of these incorporated genes has not been achieved to date. New combinations of fusion genes may be an effective way of producing transgenic domestic animals which show controlled expression of the desired genes. Embryo manipulation and genetic engineering in livestock species is moving rapidly. The problems being addressed at present in numerous laboratories will result in enhanced livestock production in the not too distant future. Key words: Embryo transfer, embryo manipulation, transgenic livestock, genetic engineering, gene transfer, monozygotic twins

GUIDELINES FOR THE REGULATION OF GENETICALLY ENGINEERED ANIMALS

2013 ◽  
Vol 25 (1) ◽  
pp. 322
Keyword(s):  
United States ◽  
Recombinant Dna ◽  
Scientific Information ◽  
The United States ◽  
Genetically Engineered ◽  
The Body ◽  
United States Government ◽  
Laboratory Animals ◽  
National Environmental Policy Act ◽  
Food Species

The FDA has been regulating genetically engineered (GE) animals under the new animal drug provisions of the Federal Food, Drug, and Cosmetic Act (FD&C Act or the Act) since producers of these animals first approached the agency in the mid-1990s, although it did not issue a final Guidance for Industry clarifying its statutory authority until 2009 (Regulation of Genetically Engineered Animals Containing Heritable rDNA Constructs: http://www.fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/UCM113903.pdf). The regulatory trigger that enables the regulation of these animals under the new animal drug provisions of the Act is the definition of a drug as “an article (other than food) that is intended to affect the structure or any function of the body of man or other animals.” The products of modern biotechnology (such as spliced recombinant DNA) are technically the “articles” that must be approved, but for shorthand, we often refer to the regulation of “genetically engineered” animals. For the purpose of the guidance, FDA defined “genetically engineered (GE) animals” as those animals modified by rDNA techniques, including all progeny that contain the modification. The term GE animal can refer both to an animal with a heritable rDNA construct or its residues and to an animal with a nonheritable rDNA construct (e.g. a construct intended as therapy for a disease in that animal). If the agency engages in an “action,” such as an approval, obligations under the National Environmental Policy Act (NEPA) are triggered, such that the agency must perform an environmental assessment to determine whether a significant impact is likely to occur on the environment of the United States. If not, the agency issues a Finding of No Significant Impact (FONSI). If a significant impact is likely to occur as the result of an agency action, the agency must engage in a more complex process to characterise that impact in an Environmental Impact Statement (EIS). This regulation is consistent with the Coordinated Framework for the Regulation of Biotechnology, a policy that was first issued by issued by the Office of Science and Technology Policy [51 Fed Reg 23,302 (1986)] of the United States Government. It describes the interagency mechanism for “sharing scientific information related to biotechnology,” and states that, to the extent possible, jurisdiction of the products of biotechnology lies with a single agency. Where more than one agency will review a particular product, “the policy establishes a lead agency and consolidated or coordinated reviews.” Consistent with this policy, FDA has leveraged, and intends to continue to leverage, the expertise of other agencies in the review of GE animal-related applications. Under certain conditions, based on risk, the agency may not enforce the premarket approval requirement for some GE animals. In general, these include GE animals of non-food species that are regulated by other government agencies or entities, such as GE animals of non-food species that are raised and used in contained and controlled conditions such as GE laboratory animals (e.g. mice, rats, some model fish) used in research institutions. In addition, on a case-by-case basis, the agency may consider exercising enforcement discretion for GE animals of very low risk, non-food species GE animals, such as the Zebra danio aquarium fish genetically engineered to fluoresce in the dark (GloFish). An exemption from the prohibition on introducing an unapproved new animal drug in interstate commerce is provided for in the regulations covering “investigations,” which allow for lawful research to occur, including the shipping of GE animals or their gametes from the sponsor of an investigation to other qualified investigators. These and other responsibilities are outlined in GFI 187, as are recommendations for the submission of data to be reviewed by CVM’s hierarchical risk-based review, and will be the subject of this talk.

Fabrication of Genetically Engineered Silk-Elastin-Like Protein Polymer Fibers

2008 ◽  
Author(s):  
Weiguo Qiu ◽  
Joseph Cappello ◽  
Xiaoyi Wu
Keyword(s):  
Tissue Engineering ◽  
Biomedical Applications ◽  
Recombinant Dna ◽  
Building Blocks ◽  
Genetically Engineered ◽  
Polymer Fibers ◽  
Structural Support ◽  
Protein Polymer ◽  
Protein Fibers ◽  
Cellular Matrices

Micro- and submicro-diameter protein fibers are fundamental building blocks of extra- and intra-cellular matrices, providing structural support, stability and protection to cells, tissues and organism [1]. Fabricating performance fibers of both naturally derived and genetically engineered proteins has been extensively pursued for a variety of biomedical applications, including tissue engineering and drug delivery [2]. Silk-elastin-like proteins (SELPs), consisting of tandemly repeated polypeptide sequences derived from silk and elastin, have been biosynthesized using recombinant DNA technique [3]. Their potential as a biomaterials in the form of hydrogels continues to be explored [4, 5]. This study will focus on the fabrication of robust, micro-diameter SELP fibers as biomaterials for tissue engineering applications.

Recombinant plasmid mobilization betweenE.colistrains in seven sterile microcosms

1997 ◽  
Vol 43 (6) ◽  
pp. 534-540 ◽  
Author(s):  
P. Lebaron ◽  
P. Bauda ◽  
N. Frank ◽  
M. C. Lett ◽  
B. Roux ◽  
...  
Keyword(s):  
Recombinant Plasmid ◽  
Recombinant Dna ◽  
Transfer Functions ◽  
Genetically Engineered ◽  
Plasmid Transfer ◽  
Transfer Rates ◽  
E Coli ◽  
Attached Biomass ◽  
Plasmid Mobilization ◽  
Dna Plasmid

Transfer by mobilization of a pBR derivative recombinant plasmid lacking transfer functions (oriT+, tra−, mob−) from one E. coli K12 strain to another was investigated in seven sterile microcosms corresponding to different environments. These microcosms were chosen as representative of environments that genetically engineered microorganisms (GEMOs) encounter after accidental release, namely attached biomass in aquatic environments (biofilm), soil, seawater, freshwater, wastewater, mouse gut, and mussel gut. GEMOs survived in the same way as the host strains in all microcosms. Recombinant DNA mobilization occurred in the mouse gut, in sterile soil, and in biofilm. The plasmid transfer rates principally reflected the environmental conditions encountered in each microcosm.Key words: recombinant DNA, plasmid transfer, mobilization, conjugation, microcosm.

scholarly journals Within-patient mutation frequencies reveal fitness costs of CpG dinucleotides and drastic amino acid changes in HIV

2016 ◽  
Author(s):  
Kristof Theys ◽  
Alison F. Feder ◽  
Maoz Gelbart ◽  
Marion Hartl ◽  
Adi Stern ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mutation Rate ◽  
Fitness Landscape ◽  
Fitness Costs ◽  
Nonsense Mutations ◽  
Cpg Dinucleotides ◽  
Synonymous Mutations ◽  
The Cost ◽  
Patient Mutation

AbstractHIV has a high mutation rate, which contributes to its ability to evolve quickly. However, we know little about the fitness costs of individual HIV mutationsin vivo, their distribution and the different factors shaping the viral fitness landscape. We calculated the mean frequency of transition mutations at 870 sites of thepolgene in 160 patients, allowing us to determine the cost of these mutations. As expected, we found high costs for non-synonymous and nonsense mutations as compared to synonymous mutations. In addition, we found that non-synonymous mutations that lead to drastic amino acid changes are twice as costly as those that do not and mutations that create new CpG dinucleotides are also twice as costly as those that do not. We also found that G→A and C→T mutations are more costly than A→G mutations. We anticipate that our newin vivofrequency-based approach will provide insights into the fitness landscape and evolvability of not only HIV, but a variety of microbes.Author summaryHIV’s high mutation rate allows it to evolve quickly. However, most mutations probably reduce the virus’ ability to replicate – they are costly to the virus. Until now, the actual cost of mutations is not well understood. We used within-patient mutation frequencies to estimate the cost of 870 HIV mutationsin vivo. As expected, we found high costs for non-synonymous and nonsense mutations. In addition, we found surprisingly high costs for mutations that lead to drastic amino acid changes, mutations that create new CpG sites (possibly because they trigger the host’s immune system), and G→A and C→T mutations. Our results demonstrate the power of analyzing mutant frequencies fromin vivoviral populations to study costs of mutations. A better understanding of fitness costs will help to predict the evolution of HIV.

scholarly journals Opportunities for Biotechnology Transfer to Developing Countries

1993 ◽  
Vol 3 (3) ◽  
pp. 268-279
Author(s):  
Don J. Durzan ◽  
M.D. Durzan
Keyword(s):  
Developing Countries ◽  
Intellectual Property ◽  
Biological Diversity ◽  
Recombinant Dna ◽  
Developing Economies ◽  
Germplasm Conservation ◽  
International Agreements ◽  
Genetically Engineered ◽  
Adaptive Plasticity ◽  
Local Risk

Prospects for the establishment of joint-ventured agribusiness in developing countries are a function of international agreements, local risk conditions, business networks, and banking systems that are willing to support the innovative transfer, protection, assessment, and commercialization of biotechnology. The integration of biotechnology will occur only if truly convincing practices emerge that enhance biodiversity and the competitiveness of sustainable production, utilization, and marketing cycles. Integration also depends on agreements on intellectual property rights, plant protection, trade and tariffs, price stabilization, and non-trade-distorting policies. These policies deal with broad issues in research, pest and disease control, environmental quality, germplasm conservation, resource retirement programs, and even with crop and disaster insurance. Measures derived from these policies will apply to novel processes and to organisms that have been genetically engineered and approved for release into the environment. For developing countries, much more attention will have to be paid to biological diversity and sustainable balances among intercropped agriforest and horticultural production systems. Balances should be compatible with regional and local customs and practices before genetically engineered “green goods and services” are introduced in the marketplace. Recombinant DNA technologies are currently better-suited to deal on a “gene-by-gene” basis, with commodity surpluses and material conversions involving more concentrated and industrialized processes than with field plantations of genetically engineered, complex, and long-lived crops that may require considerable adaptive plasticity. In most countries with developing economies, the integration of recombinant DNA technology represents a “special problematique” involving politico- and socioeconomic and environmental factors. Barriers to transfer and integration may involve evolving international agreements, public acceptance, resource over-exploitation, environmental degradation, rapid insect and disease resistance, contaminated water and food supplies, reduced quality of life, labor quality, corruption, crime, farmers' rights, germplasm conservation, and lack of protection of intellectual property, among other factors. Hence, the timing and mode of transferring biotechnology needs considerable impact assessment on a case-by-case basis.

scholarly journals Escherichia coli with a Tunable Point Mutation Rate for Evolution Experiments

2020 ◽  
Vol 10 (8) ◽  
pp. 2671-2681 ◽  
Author(s):  
Nicholas A. Sherer ◽  
Thomas E. Kuhlman
Keyword(s):  
Escherichia Coli ◽  
Point Mutation ◽  
Mutation Rate ◽  
Fitness Landscape ◽  
Genetically Engineered ◽  
Mutation Rates ◽  
Nucleotide Polymorphisms ◽  
Beneficial Mutations ◽  
Beneficial Effects ◽  
Evolution Experiment

The mutation rate and mutations’ effects on fitness are crucial to evolution. Mutation rates are under selection due to linkage between mutation rate modifiers and mutations’ effects on fitness. The linkage between a higher mutation rate and more beneficial mutations selects for higher mutation rates, while the linkage between a higher mutation rate and more deleterious mutations selects for lower mutation rates. The net direction of selection on mutations rates depends on the fitness landscape, and a great deal of work has elucidated the fitness landscapes of mutations. However, tests of the effect of varying a mutation rate on evolution in a single organism in a single environment have been difficult. This has been studied using strains of antimutators and mutators, but these strains may differ in additional ways and typically do not allow for continuous variation of the mutation rate. To help investigate the effects of the mutation rate on evolution, we have genetically engineered a strain of Escherichia coli with a point mutation rate that can be smoothly varied over two orders of magnitude. We did this by engineering a strain with inducible control of the mismatch repair proteins MutH and MutL. We used this strain in an approximately 350 generation evolution experiment with controlled variation of the mutation rate. We confirmed the construct and the mutation rate were stable over this time. Sequencing evolved strains revealed a higher number of single nucleotide polymorphisms at higher mutations rates, likely due to either the beneficial effects of these mutations or their linkage to beneficial mutations.

SUSTAINED EXPRESSION OF FULL LENGTH AND VARIANT RECOMBINANT FACTOR VIII IN GENETICALLY ENGINEERED CELLS

1987 ◽  
Author(s):  
Nava Sarver ◽  
George A Ricca
Keyword(s):  
Factor Viii ◽  
Recombinant Dna ◽  
Expression System ◽  
Genetically Engineered ◽  
Full Length ◽  
Cdna Insert ◽  
Mammalian Expression ◽  
Coagulant Activity ◽  
Recombinant Fviii

A major effort is presently underway to provide factor VIII (FVIII) in a form free of viral pathogens via a recombinant DNA approach. We have constructed two chimeric FVIII cDNA vectors based on the bovine papillomavirus mammalian expression system. The first vector (FVIII) contained a full length FVIII cDNA; the second vector (AFVIII) contained a cDNA insert with an extensive deletion, corresponding to amino acid residues 747 to 1560 in the region encoding the "B" domain. This internal region is removed during activation of the parental FVIII molecule and is believed not to be required for coagulant activity. We have found that recombinant FVIII produced by stable cell lines harboring either the full length or the variant FVIII was capable of restoring coagulant activity to FVIII deficient plasma in. vitro. This expressed activity was neutralized by anti-FVIII antibodies. Similar to observations with FVIII derived from human plasma, the two recombinant FVIII forms were (i) inactivated by the chelating agent EDTA, (ii) demonstrated a biphasic response of an initial activation followed by a decay in activity when treated with thrombin, and (iii) presented the expected peptide banding pattern by western blot analyses. A higher percentage of ΔFVIII transformants were isolated expressing coagulant activity compared to transformants harboring the complete FVIII cDNA. Among the positive transformants isolated, those harboring ΔFVIII produced higher levels of coagulant activity than their full length counterparts. Comparable steady state levels of FVIII specific transcripts were detected in FVIII and ΔFVIII transformants indicating that the difference in expression levels is due to a post transcriptional event(s). Our study demonstrates the efficacy of a full length and an abridged recombinant FVIII produced by stably transformed cells in correcting coagulation deficiency in. vitro. It further suggests the potential usefulness of other molecular variants for efficient expression in genetically engineered cells.

Transgenic Livestock in Agriculture: New Animals for a New World?

1995 ◽  
Vol 24 (4) ◽  
pp. 227-232
Author(s):  
Caird E. Rexroad
Keyword(s):  
New World ◽  
Recombinant Dna ◽  
Transgenic Animals ◽  
Genetically Engineered ◽  
Recombinant Dna Technology ◽  
Gene Insertion ◽  
New Genes ◽  
Transgenic Livestock ◽  
Dna Technology

Recombinant DNA technology and techniques for gene insertion into animals have generated promises of animals genetically engineered to be healthy, productive, and sources of novel and/or improved foods and fibre, Transgenic animals have also generated concerns about the production of monsters that will cause ecological catastrophes. Here, current research on the insertion of new genes into livestock is described, and an attempt is made to provide a scientific perspective on the likelihood of either outcome.

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