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CLONING IN EUKARYOTES
SACCHAROMYCES CEREVISIAE
PICHIA PASTORIS
By
Priyengha R S
GENE CLONING
 Gene cloning is the process of incorporating
foreign genes into hybrid DNA replicons.
 Cloned genes can be expressed in appropriate
host cells, and the phenotypes that they determine
can be analyzed.
 A Clone is a population of organisms or molecules
derived by asexual reproduction from a single
ancestor.
 Some key concepts underlying representative
methods are summarized here.
DNA CLONING
 Restriction enzymes (endonucleases):
in nature, these enzymes
protect bacteria from intruding DNA; they
cut up the DNA (restriction); very specific
 Restriction site:
recognition sequence for a
particular restriction enzyme
 Restriction fragments:
segments of DNA cut by
restriction enzymes in a reproducable
way
 Sticky end:
short extensions of
restriction fragments
 DNA ligase:
enzyme that can join the
sticky ends of DNA fragments
 Cloning vector:
DNA molecule that can carry
foreign DNA into a cell and replicate there
(usually bacterial plasmids)
STEPS FOR EUKARYOTIC GENE
CLONING
 Isolation of cloning vector
(bacterial plasmid) & gene-
source DNA (gene of interest)
 Insertion of gene-source DNA
into the cloning vector using the
same restriction enzyme; bind
the fragmented DNA with DNA
ligase
 Introduction of cloning vector into
cells (transformation by bacterial
cells)
 Cloning of cells (and foreign
genes)
 Identification of cell clones
carrying the gene of interest
Restriction
endonucleases
WHY CLONE IN EUKARYOTES?
 Eukaryotic genes may not be expressed properly in
bacterial host
 different mechanisms for gene expression
 modifications (glycosylation)
 very large pieces of DNA can be cloned (yACs)
Cloning in S. cerevisiae
(cloning in eukaryotes)
Chapter11GeneticEngineering:Gene
Cloning
TRANSFECTION OF
EUKARYOTIC CELLS
Transfection originally referred to
transformation of viral nucleic acid into cells
(both bacterial and eukaryotic)
 Microinjection- a technique where DNA can
also be injected directly into the nucleus using
micropipets
 Electroporation- exposes host cells to pulsed
electrical fields in the presence of cloned DNA.
 Particle gun or gene gun- high-velocity
microprojectile “gun” can be used to get DNA
into cells.
SHUTTLE VECTORS AND EXPRESSION
VECTORS
 Shuttle Vectors
 Vectors that can replicate and are stably
maintained in two (or more) unrelated host
organisms
 Genes carried these vectors can be moved
between unrelated organisms. Have been
developed that replicate in both Escherichia
coli and Bacillus subtilis
 Convenient marker to select for the plasmid
in yeast
 Importance: DNA cloned in one organism
can be replicated in a second host without
modifying the vector
Genetic map of a shuttle vector
used in yeast
Expression Vectors
 are designed to allow the experimenter to
control the expression of cloned
 contain regulatory sequences that allow
manipulation of gene expression
 Promoters from E. coli that are used in
expression vectors include lac (the lac operon
promoter), trp (the trp operon promoter), tac
and trc (synthetic hybrids of the trp and lac
promoters), and lambda PL (the leftward
lambda promoter;
Genetic map of the expression
vector pSE420.
EUKARYOTIC VECTORS
It is often desirable to clone and express
genes directly in eukaryotes, and vectors
are available for cloning into yeast
Two-micron circle- A plasmid where most
yeast vectors are based on this
Virus vectors are commonly used in
multicellular eukaryotes.
Integrating vectors
are maintained at low copy number by
integrating into the host chromosome
BACTERIOPHAGE LAMBDA AS A
CLONING VECTOR
Lambda can also be used as a cloning
vector for in vitro recombination
useful cloning vector because it can hold
larger amounts of DNA than most
plasmids and DNA can be efficiently
packaged into phage particles in vitro.
Bacteriophage lambda- most studied of
the specialized transducing phages
Phage lambda- has a large number of
genes.
MODIFIED LAMBDA PHAGES
Wild-type lambda is not suitable as a
cloning vector because its genome has too
many restriction enzyme sites
Charon phages- one set of modified
lambda phages
unwanted restriction enzyme sites have
been removed by mutation.
Replacement vectors are especially
useful in cloning large DNA fragments
EUKARYOTIC ARTIFICIAL CHROMOSOMES
 Yeast Artificial Chromosomes (YACs)
 first artificial chromosomes
 replicate in yeast like normal chromosomes, but they
have sites where very large fragments of DNA can be
inserted.
 To function like normal eukaryotic chromosomes
(1) an origin of DNA replication,
(2) telomeres for replicating DNA at the ends of the
chromosome and
(3) a centromere for segregation during mitosis.
(4) cloning site
(5) gene
for selection transformation
into the host
Human artificial chromosomes (HACs)
Have also been developed and are
similar to YACs in overall structure.
Circular*
Have long arrays of these repeats to be
inherited
Must be stably
Mammalian centromere consists of long
stretches of repeated sequences and
HACs.
• The choice of an expression system depends
primarily on the quality of the recombinant protein.
• However, the yield of the product and cost of
production and purification are also important
considerations.
• The vector must be designed to be maintained in
the eukaryotic host.
• The vector must have eukaryotic promoter,
transcriptional, translational stop signals, a
sequence that enables polyadenylation and a
selectable marker gene.
EUKARYOTIC EXPRESSION SYSTEMS
• The major features of a eukaryotic expression vector
are a promoter, a multiple cloning site, DNA segment
for termination and polyadenylation, selectable marker,
origin of replication in E. coli and eukaryotic cell and
Ampr for marker in E. coli.
• Saccharomyces cerevisiae
• Pichia pastoris
• Baculovirus-insect cell lines
• Mammalian systems
EUKARYOTIC EXPRESSION SYSTEMS
• It is the most common eukaryotic system and
there is a great deal of study about this organism.
• It is a single-celled and behaves like a bacterial
culture and can be grown in relatively simple
media in both small and large-scale production.
• Well characterized with many strong regulatable
promoters with naturally occurring plasmids.
• Carry out post-translational modifications.
• Secretes very few of its own proteins.
• Recognized as safe by USDA and FDA.
SACCHAROMYCES CEREVISIAE
Cloning in eukaryotes
• There are three main classes of S. cerevisiae
expression vectors.
• Yeast episomal plasmids (YEps).
• Yeast integrating plasmids (YIps)
• Yeast artificial chromosomes (YACs)
• Yeast episomal plasmids have been used
extensively for the production of eitehr intra- or
extracellular heterologous proteins.
• Typically, vectors function in both E. coli and S.
cerevisiae.
SACCHAROMYCES CEREVISIAE
• The YEps vectors are based on the high-copy-
number 2µm plasmids.
• The vectors replicate independently via a single
origin of replication.
• There are more than 30 copies per cell.
• Selection scheme rely on mutant host strains
that require a particular amino acid (histidine,
tryptophan, or leucine) or nucleotide (uracil).
• When a Yep with a wild-type LEU2 gene is
transformed into a mutant leu2 host cell, only
cells that carry plasmid will grow.
SACCHAROMYCES CEREVISIAE
• Generally, tightly regulatable, inducible promoters are
preferred for producing large amounts of recombinant
protein at a specific time during large-scale growth.
• Most heterologous genes are provided with a
DNA coding sequence for signal peptide that
facilitates the secretion of protein through cell
membranes and external environment.
• Other sequence that protect the recombinant
protein from proteolytic degradation, and provide
a affinity tag is also used.
• These extra amino acid sequences are equipped
with a protease cleavage site so that they can be
removed from the recombinant protein.
SACCHAROMYCES CEREVISIAE
• Plasmid-based yeast expression systems are often
unstable under large-scale growth conditions even
in the presence of selection pressure.
• A Yip vector is used to integrate a heterologous
gene into the host genome to provide a more
reliable production system.
• The plasmid does not usually carry an origin of
replication.
• The disadvantage is the low yield of recombinant
protein from a single gene copy.
SACCHAROMYCES CEREVISIAE
Integration of DNA with a Yip vector
YEAST ARTIFICIAL CHROMOSOMES
 YAC is an artificially constructed chromosome that
contains a
 Centromere
 Telomeres
 Autonomous replicating sequence (ARS) element
required for replication and preservation of YAC
in yeast cells
 ARS elements are thought to act as replication origins
 First described in 1983 by Murray and Szostak
YACs are plasmid shuttle vectors capable of
replicating and being selected in common
bacterial hosts such as Escherichia coli, as
well as in the budding yeast
Saccharomyces cerevisiae.
Yeast artificial chromosome (YAC) is
a human-engineered DNA molecule
used to clone DNA sequences in
yeast cells
Purpose of using YAC Vectors:
Cloning vehicles that propogate in eukaryotic cell hosts as
eukaryotic Chromosomes
Clone very large inserts of DNA: 100 kb - 10 Mb
Features:
YAC cloning vehicles are plasmids
Final chimeric DNA is a linear DNA molecule with telomeric
ends: Artificial Chromosome
CONSTRUCTION OF YEAST ARTIFICIAL
CHROMOSOMES
Plasmid DNA purification
Treatment with restriction
enzymes
Ligation and yeast
transformation
Cloning in eukaryotes
• A YAC is designed to clone a large segment of DNA
(100 kb), which is then maintained as a separate
chromosome in the host yeast cell.
• It is highly stable and has been used for the physical
mapping of human genomic DNA, the analysis of
transcription units, and genomic libraries.
• It has a sequences that act as ARS for replication,
centromere for cell division, and telomere for stability.
• To date, they have not been used as expression
systems for the commercial production.
YAC cloning system
YAC cloning system
• Human Cu/Zn SOD cDNA was cloned between the
promoter and termination-polyadenylation sequence of
the yeast GAPD gene and subsequently used to
transform LEU- mutant host cell.
Intercellular Production in Yeast
• Proteins may also be produced for secretion.
• In this system, any glycosylated protein is secreted (O
or N-linked).
• The coding sequences of recombinant proteins must be
cloned downstream of a leader sequence, the yeast
mating type factor α-factor.
• Under these conditions, correct disulfide bond
formation, proteolytic removal of the leader sequence,
and appropriate posttranslational modifications occur,
and an active recombinant protein is secreted.
• The leader peptide is removed by endoprotease that
recognizes the Lys-Arg.
Secretion of Heterologous Proteins
• For example, a properly processed and active form of
the protein hirudin; a powerful anticoagulant protein
cloned from a leech, was synthesized and secreted by
an S. cerevisiae.
• A YEp vector that had the prepro-α-factor sequence
added to the huridin coding sequencea to allow
expression that is cleaved away in processing.
• Leaves active hirudin which is secreted.
• Producing a recombinant protein for use in human
therapeutics in yeast rather than in bacteria is to ensure
the proper folding.
Secretion of Heterologous Proteins
Secretion of Heterologous Proteins
• Though S. cerevisae is successfully used to produce
recombinant proteins for human, it has major
drawbacks.
• The level of protein production is low.
• There is the tendency for hyperglycosylation resulting
in change of protein function.
• Proteins are often retained in periplasm, increasing
time and cost for purification.
• It produces ethanol at high cell densities, which is toxic
to cells.
PICHIA PASTORIS EXPRESSION SYSTEMS
• P. pastoris is a methylotrophic yeast that is able to
utilize methanol as a source of carbon and energy.
• Glycosylation occurs to a lesser extent and the
linkages between sugar residues are of the α-1,2 type.
• P. pastoris strain was extensively engineered with the
aim of developing a “humanized” strain that glycosylate
proteins in a manner identical to that of human cells.
• It does not produce ethanol.
• It normally secretes very few proteins, thus simplifying
the purification of secreted recombinant proteins.
PICHIA PASTORIS EXPRESSION SYSTEMS
Pichia pastoris Expression Systems
Pichia pastoris Expression Systems
Pichia pastoris vs Saccharomyces cerevisiae
Advantages P. pastoris and S. cerevisiae
Short doubling time
Readily manipulated genome
Improved folding and post-translational modification
Expression of similar genes and compatible vectors
Better yield of recombinant protein (higher cell density)
Methylotrophic yeast (methanol as its only carbon source)
Strongly methanol induced promoters
(alcohol oxidase genes: AOX1 and AOX2)
Optimal growth pH 3.0-7.0
Extremely low levels of endogenous protein secretion
Expression vectors integrated in the genome
Disulfide bond formation and glycosylation modifications
S. cerevisiae
P. pastoris
PRACTICAL DNA TECHNOLOGY USES
 Diagnosis of disease
 Human gene therapy
 Pharmaceutical products
(vaccines)
 Forensics
 Animal husbandry
(transgenic organisms)
 Genetic engineering in
plants
 Ethical concerns?
GENES THERAPY
BIOTECHNOLOGY PRACTICAL USE
Thank You..

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Cloning in eukaryotes

  • 1. CLONING IN EUKARYOTES SACCHAROMYCES CEREVISIAE PICHIA PASTORIS By Priyengha R S
  • 2. GENE CLONING  Gene cloning is the process of incorporating foreign genes into hybrid DNA replicons.  Cloned genes can be expressed in appropriate host cells, and the phenotypes that they determine can be analyzed.  A Clone is a population of organisms or molecules derived by asexual reproduction from a single ancestor.  Some key concepts underlying representative methods are summarized here.
  • 3. DNA CLONING  Restriction enzymes (endonucleases): in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific  Restriction site: recognition sequence for a particular restriction enzyme  Restriction fragments: segments of DNA cut by restriction enzymes in a reproducable way  Sticky end: short extensions of restriction fragments  DNA ligase: enzyme that can join the sticky ends of DNA fragments  Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)
  • 4. STEPS FOR EUKARYOTIC GENE CLONING  Isolation of cloning vector (bacterial plasmid) & gene- source DNA (gene of interest)  Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase  Introduction of cloning vector into cells (transformation by bacterial cells)  Cloning of cells (and foreign genes)  Identification of cell clones carrying the gene of interest
  • 6. WHY CLONE IN EUKARYOTES?  Eukaryotic genes may not be expressed properly in bacterial host  different mechanisms for gene expression  modifications (glycosylation)  very large pieces of DNA can be cloned (yACs) Cloning in S. cerevisiae (cloning in eukaryotes)
  • 8. TRANSFECTION OF EUKARYOTIC CELLS Transfection originally referred to transformation of viral nucleic acid into cells (both bacterial and eukaryotic)  Microinjection- a technique where DNA can also be injected directly into the nucleus using micropipets  Electroporation- exposes host cells to pulsed electrical fields in the presence of cloned DNA.  Particle gun or gene gun- high-velocity microprojectile “gun” can be used to get DNA into cells.
  • 9. SHUTTLE VECTORS AND EXPRESSION VECTORS  Shuttle Vectors  Vectors that can replicate and are stably maintained in two (or more) unrelated host organisms  Genes carried these vectors can be moved between unrelated organisms. Have been developed that replicate in both Escherichia coli and Bacillus subtilis  Convenient marker to select for the plasmid in yeast  Importance: DNA cloned in one organism can be replicated in a second host without modifying the vector
  • 10. Genetic map of a shuttle vector used in yeast
  • 11. Expression Vectors  are designed to allow the experimenter to control the expression of cloned  contain regulatory sequences that allow manipulation of gene expression  Promoters from E. coli that are used in expression vectors include lac (the lac operon promoter), trp (the trp operon promoter), tac and trc (synthetic hybrids of the trp and lac promoters), and lambda PL (the leftward lambda promoter;
  • 12. Genetic map of the expression vector pSE420.
  • 13. EUKARYOTIC VECTORS It is often desirable to clone and express genes directly in eukaryotes, and vectors are available for cloning into yeast Two-micron circle- A plasmid where most yeast vectors are based on this Virus vectors are commonly used in multicellular eukaryotes. Integrating vectors are maintained at low copy number by integrating into the host chromosome
  • 14. BACTERIOPHAGE LAMBDA AS A CLONING VECTOR Lambda can also be used as a cloning vector for in vitro recombination useful cloning vector because it can hold larger amounts of DNA than most plasmids and DNA can be efficiently packaged into phage particles in vitro. Bacteriophage lambda- most studied of the specialized transducing phages Phage lambda- has a large number of genes.
  • 15. MODIFIED LAMBDA PHAGES Wild-type lambda is not suitable as a cloning vector because its genome has too many restriction enzyme sites Charon phages- one set of modified lambda phages unwanted restriction enzyme sites have been removed by mutation. Replacement vectors are especially useful in cloning large DNA fragments
  • 16. EUKARYOTIC ARTIFICIAL CHROMOSOMES  Yeast Artificial Chromosomes (YACs)  first artificial chromosomes  replicate in yeast like normal chromosomes, but they have sites where very large fragments of DNA can be inserted.  To function like normal eukaryotic chromosomes (1) an origin of DNA replication, (2) telomeres for replicating DNA at the ends of the chromosome and (3) a centromere for segregation during mitosis. (4) cloning site (5) gene for selection transformation into the host
  • 17. Human artificial chromosomes (HACs) Have also been developed and are similar to YACs in overall structure. Circular* Have long arrays of these repeats to be inherited Must be stably Mammalian centromere consists of long stretches of repeated sequences and HACs.
  • 18. • The choice of an expression system depends primarily on the quality of the recombinant protein. • However, the yield of the product and cost of production and purification are also important considerations. • The vector must be designed to be maintained in the eukaryotic host. • The vector must have eukaryotic promoter, transcriptional, translational stop signals, a sequence that enables polyadenylation and a selectable marker gene. EUKARYOTIC EXPRESSION SYSTEMS
  • 19. • The major features of a eukaryotic expression vector are a promoter, a multiple cloning site, DNA segment for termination and polyadenylation, selectable marker, origin of replication in E. coli and eukaryotic cell and Ampr for marker in E. coli.
  • 20. • Saccharomyces cerevisiae • Pichia pastoris • Baculovirus-insect cell lines • Mammalian systems EUKARYOTIC EXPRESSION SYSTEMS
  • 21. • It is the most common eukaryotic system and there is a great deal of study about this organism. • It is a single-celled and behaves like a bacterial culture and can be grown in relatively simple media in both small and large-scale production. • Well characterized with many strong regulatable promoters with naturally occurring plasmids. • Carry out post-translational modifications. • Secretes very few of its own proteins. • Recognized as safe by USDA and FDA. SACCHAROMYCES CEREVISIAE
  • 23. • There are three main classes of S. cerevisiae expression vectors. • Yeast episomal plasmids (YEps). • Yeast integrating plasmids (YIps) • Yeast artificial chromosomes (YACs) • Yeast episomal plasmids have been used extensively for the production of eitehr intra- or extracellular heterologous proteins. • Typically, vectors function in both E. coli and S. cerevisiae. SACCHAROMYCES CEREVISIAE
  • 24. • The YEps vectors are based on the high-copy- number 2µm plasmids. • The vectors replicate independently via a single origin of replication. • There are more than 30 copies per cell. • Selection scheme rely on mutant host strains that require a particular amino acid (histidine, tryptophan, or leucine) or nucleotide (uracil). • When a Yep with a wild-type LEU2 gene is transformed into a mutant leu2 host cell, only cells that carry plasmid will grow. SACCHAROMYCES CEREVISIAE
  • 25. • Generally, tightly regulatable, inducible promoters are preferred for producing large amounts of recombinant protein at a specific time during large-scale growth.
  • 26. • Most heterologous genes are provided with a DNA coding sequence for signal peptide that facilitates the secretion of protein through cell membranes and external environment. • Other sequence that protect the recombinant protein from proteolytic degradation, and provide a affinity tag is also used. • These extra amino acid sequences are equipped with a protease cleavage site so that they can be removed from the recombinant protein. SACCHAROMYCES CEREVISIAE
  • 27. • Plasmid-based yeast expression systems are often unstable under large-scale growth conditions even in the presence of selection pressure. • A Yip vector is used to integrate a heterologous gene into the host genome to provide a more reliable production system. • The plasmid does not usually carry an origin of replication. • The disadvantage is the low yield of recombinant protein from a single gene copy. SACCHAROMYCES CEREVISIAE
  • 28. Integration of DNA with a Yip vector
  • 29. YEAST ARTIFICIAL CHROMOSOMES  YAC is an artificially constructed chromosome that contains a  Centromere  Telomeres  Autonomous replicating sequence (ARS) element required for replication and preservation of YAC in yeast cells  ARS elements are thought to act as replication origins  First described in 1983 by Murray and Szostak
  • 30. YACs are plasmid shuttle vectors capable of replicating and being selected in common bacterial hosts such as Escherichia coli, as well as in the budding yeast Saccharomyces cerevisiae. Yeast artificial chromosome (YAC) is a human-engineered DNA molecule used to clone DNA sequences in yeast cells
  • 31. Purpose of using YAC Vectors: Cloning vehicles that propogate in eukaryotic cell hosts as eukaryotic Chromosomes Clone very large inserts of DNA: 100 kb - 10 Mb Features: YAC cloning vehicles are plasmids Final chimeric DNA is a linear DNA molecule with telomeric ends: Artificial Chromosome
  • 32. CONSTRUCTION OF YEAST ARTIFICIAL CHROMOSOMES Plasmid DNA purification Treatment with restriction enzymes Ligation and yeast transformation
  • 34. • A YAC is designed to clone a large segment of DNA (100 kb), which is then maintained as a separate chromosome in the host yeast cell. • It is highly stable and has been used for the physical mapping of human genomic DNA, the analysis of transcription units, and genomic libraries. • It has a sequences that act as ARS for replication, centromere for cell division, and telomere for stability. • To date, they have not been used as expression systems for the commercial production. YAC cloning system
  • 36. • Human Cu/Zn SOD cDNA was cloned between the promoter and termination-polyadenylation sequence of the yeast GAPD gene and subsequently used to transform LEU- mutant host cell. Intercellular Production in Yeast
  • 37. • Proteins may also be produced for secretion. • In this system, any glycosylated protein is secreted (O or N-linked). • The coding sequences of recombinant proteins must be cloned downstream of a leader sequence, the yeast mating type factor α-factor. • Under these conditions, correct disulfide bond formation, proteolytic removal of the leader sequence, and appropriate posttranslational modifications occur, and an active recombinant protein is secreted. • The leader peptide is removed by endoprotease that recognizes the Lys-Arg. Secretion of Heterologous Proteins
  • 38. • For example, a properly processed and active form of the protein hirudin; a powerful anticoagulant protein cloned from a leech, was synthesized and secreted by an S. cerevisiae. • A YEp vector that had the prepro-α-factor sequence added to the huridin coding sequencea to allow expression that is cleaved away in processing. • Leaves active hirudin which is secreted. • Producing a recombinant protein for use in human therapeutics in yeast rather than in bacteria is to ensure the proper folding. Secretion of Heterologous Proteins
  • 40. • Though S. cerevisae is successfully used to produce recombinant proteins for human, it has major drawbacks. • The level of protein production is low. • There is the tendency for hyperglycosylation resulting in change of protein function. • Proteins are often retained in periplasm, increasing time and cost for purification. • It produces ethanol at high cell densities, which is toxic to cells. PICHIA PASTORIS EXPRESSION SYSTEMS
  • 41. • P. pastoris is a methylotrophic yeast that is able to utilize methanol as a source of carbon and energy. • Glycosylation occurs to a lesser extent and the linkages between sugar residues are of the α-1,2 type. • P. pastoris strain was extensively engineered with the aim of developing a “humanized” strain that glycosylate proteins in a manner identical to that of human cells. • It does not produce ethanol. • It normally secretes very few proteins, thus simplifying the purification of secreted recombinant proteins. PICHIA PASTORIS EXPRESSION SYSTEMS
  • 44. Pichia pastoris vs Saccharomyces cerevisiae Advantages P. pastoris and S. cerevisiae Short doubling time Readily manipulated genome Improved folding and post-translational modification Expression of similar genes and compatible vectors Better yield of recombinant protein (higher cell density) Methylotrophic yeast (methanol as its only carbon source) Strongly methanol induced promoters (alcohol oxidase genes: AOX1 and AOX2) Optimal growth pH 3.0-7.0 Extremely low levels of endogenous protein secretion Expression vectors integrated in the genome Disulfide bond formation and glycosylation modifications S. cerevisiae P. pastoris
  • 45. PRACTICAL DNA TECHNOLOGY USES  Diagnosis of disease  Human gene therapy  Pharmaceutical products (vaccines)  Forensics  Animal husbandry (transgenic organisms)  Genetic engineering in plants  Ethical concerns?