Conclusions

1
Transient Expression of Therapeutic Proteins and
Oxygen Transport Limitations in Tissue
Culture Amalie Tuerk Department of Chemical
Engineering Dr. Wayne Curtis, Advisor
Methods Transgenic Nicotiana benthamiana plants
were created by insertion of a viral replicase
gene (REP) expressed under the control of an
ethanol inducible promoter and with an associated
kanamycin resistance gene. Replicase gene
insertion was verified by PCR. Hemi- and
homozygous plants were generated by successive
selfing and growth under kanamycin selection
pressure. Two constructs of the REP gene were
used D-intron REP (DR) was generated in hopes of
coding for a less toxic form of the REP protein
than Native-REP (RR, RF). Wild type REP (-),
hemizygous REP (), and homozygous REP () seeds
were germinated in 50 ml of liquid B5 media in
150-ml Erlenmeyer flasks on a gyratory shaker
(1.52 cm stroke, 120-150 rpm) after surface
sterilization with 10 bleach for 10 minutes.
Ambient air or oxygen-enriched air was introduced
into the headspace of the flask after being
passed through a 0.2 mm sterilization
filter. Segregation tests were conducted by
germinating seeds on solid B5 media with 300 mg/L
kanamycin as selection pressure. Seedlings that
that survived on the selection media versus
seedlings that showed bleaching were counted.
The lengths of hypocotyl and root segments were
measured by scanning seedlings on a
flat-bed scanner with a reference
scale, then digitizing length using the
NIH Image J image analysis program. Data
analysis was completed with Excel.
Objective Use segregation testing to verify
good- and poor-growing genotypes to determine if
hypocotyl length under oxygen-limited conditions
can be used to predict for the presence of REP.
The presence of REP (/-) was correctly
predicted in 12 out of 12 seedlings. The
genotype (hemi/homozygous) was correctly
predicted in 8 out of 12 seedlings.
Background and Introduction This project is part
of a larger effort to inexpensively produce
therapeutic drugs and proteins through transient
expression in plant tissue culture. This is
accomplished by co-culturing plant tissue with
Agrobacterium, a plant pathogen with the ability
to transfer DNA to plants. A viral replicase
gene (REP) was genetically engineered into plants
in order to amplify expression of the inserted
DNA. However, there was no way to functionally
demonstrate its expression because there are no
antibodies available for a traditional assay such
as a Western Blot. While exploring the
possibility of using seeds from REP() Nicotiana
benthamiana plants as a gene expression host for
co-culture, several UNEXPECTED OBSERVATIONS were
made. Seeds from plants known to be
homozygous for REP all exhibited a poor-growing
phenotype. Seeds from plants known to be
hemizygous for REP demonstrated reverse
Mendelian segregation, segregating into good- and
poor-growing phenotypes. Wild-type seeds, grown
under the same conditions, produced healthy
seedlings. Based on the observations of reverse
segregation, a series of experiments were carried
out to test the discovery of this
Metabolic-Genetic Screen based on the following
hypothesis
Example of a segregation test. Red dots indicate
bleached seedlings on selection media.
Oxygen Transport Limitation Based on mass
transfer theory, it was calculated that oxygen
transport for seeds in this system is limited by
the oxygen transport rate (OTR) from the liquid
to the seed surface (Oxygen Transport in Plant
Tissue Culture Systems Curtis, W. R. and A. L.
Tuerk 2005).
BOD ks A (CL Cs)
Objective Quantify reverse segregation of
transgenic and wild type seeds grown under
oxygen-limited and enhanced oxygen conditions.
Wild type seeds and homozygous
Native-REP() seeds demonstrated 100 good and
poor-growing phenotypes under oxygen limitation,
respectively. Hemizygous native-REP() seeds
demonstrated reverse Mendelian segregation.
The poor-growing seedlings were found to survive
on kanamycin selection media, suggesting that the
poor-growers contain the REP gene. Good-growing
seedlings bleached and died on kanamycin
selection media, indicating that these are
without a copy of the REP gene. Hemizygous
D-REP() seeds demonstrated segregation of this
less toxic REP that did not follow typical
Mendelian segregation. Some good growers
survived on kanamycin selection media, suggesting
presence of the linked REP gene.
For a Biological Oxygen Demand (BOD) of 100 mmol/
g. fresh weight seeds/ hr., the following values
were calculated At ambient conditions, OTR
is not sufficient to meet BOD. At an elevated
O2 level of 37, OTR is sufficient to meet BOD.
CENTRAL HYPOTHESIS Oxygen transport limitation
that occurs during the liquid culture of
seedlings causes anaerobic respiration in the
germinating seeds. The ethanol by-product
subsequently induces the gene coding for
the toxic protein REP, leading
to the observed poor-growing phenotype.

• Conclusions
• This research provides a demonstration of
  functional REP expression towards an
  application in amplifying gene expression.
• Germination of transgenic seedlings under
  anaerobic metabolism produces ethanol as
  a by-product, which
  induces the REP gene under the ethanol-inducible
  promoter.
• Anaerobic respiration was confirmed as the
  metabolic basis underlying the Metabolic-Genetic
  screen by reverting phenotypes under elevated
  oxygen.

• Inhibition of hypocotyl extension correlates
  with a REP() genotype and was successfully used
  to predict for the presence of REP. Prediction
  of gene dosage was less successful and is
  dependent on gene insertion effects.
• Future Research
• Show that growing seedlings under aerobic
  conditions allows for successful participation in
  co-culture and transient expression.
• Use manipulation of oxygen levels in bioreactors
  to control gene expression.

Objective Quantify hypocotyl and root extension
lengths characteristic of good- and poor-growers
and compare to wild type growth.
Hypocotyl length of poor growers is significantly
stunted in comparison to good-growers and wild
type seedlings. Growth of transgenic seedlings
under elevated oxygen conditions results in
hypocotyl lengths that are much closer to the
lengths of wild type hypocotyls, although some
stunting of growth does occur.

• Acknowledgements and Funding Sources
• National Science Foundation (REU supplement to
  Grant BCS-0003926 GOALI program)
• Research Experience for Undergraduates Program
  (Grant EEC-0353569)
• Grant from the Department of Chemical Engineering
  Endowment Fund
• Many thanks to Lauren Andrews, Kristin ONeill,
  Jason Collens, Randhir Shetty, Brandon Curtis,
  and Dr. Wayne Curtis

Definitions Homozygous Parent plant has two
copies of the REP gene. Selfing produces seeds
that are all homozygous and have two copies of
the REP gene. Hemizygous Parent plant has one
copy of the REP gene. Selfing theoretically
produces a mix of genotypes 25 homozygous, 50
hemizygous, 25 with no copy of the REP gene.