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The present status of alternatives to transgenic technology for medical research

This report presents an overview of the current status of alternatives to transgenics in

medical research. Gene silencing by ribozymes and antisense oligonucleotides, cell culture,
episomal expression vectors, and growth factor treatment are reviewed as alternatives to the
transgenic technology. Comment [N1]: The introduction gives no
clear rationale for analysing the topic. There is
no definition of the term transgenics and no
Ribozymes are RNA enzyme molecules that can cleave specific messenger RNA discussion of why alternatives to transgenics
are being sought.
(mRNA) sequences. Ribozymes contain variable sequences that determine the specificity of the
ribozyme for its target molecule and highly conserved sequences that direct catalytic hydrolysis
of the mRNA at specific trinucleotide motifs. Mutation-specific cleavage of the transcript
functionally ''silences'' the mutant allele by preventing synthesis of the abnormal protein from the
transcript (Chaum, 2002). The strategy used is to construct ribozymes that identify unique Comment [N2]: This paper attempts to use
the author-date citation system, as required for
mutations or that permit binding to targeted, accessible sites in the mRNA transcript (Chaum, a previous assignment. In that system, the in-
text citation should name both authors: ''Chaum
2002). Ribozymes are synthesized in situ from vectors, and each molecule can catalyze the & Hatton, 2002''. In the ICMJE citation-
hydrolysis of many transcripts within the cell (Chaum, 2002). Recently, a mutation-independent sequence system required this year, the in-text
citation would be simply ''(1)''. In some journals
approach to ribozyme therapy has been used in which both mutant and wild-type transcripts are it would be ''[1]''. See the final comment below
for the matching Reference entry.
cleaved but a modified wild-type transcript encoding the normal protein is introduced (Chaum,
Comment [N3]: The term in situ, like in
2002). These modified transcripts are not cleaved by the ribozyme and, thus, permit translation vitro and in vivo, should be in italics.
of the wild-type protein in the cell. Ribozymes can also be directed against wild-type alleles. Comment [N4]: The purpose of this new
approach needs to be discussed.
Thus, ribozymes can be used to silence wild-type genes to generate in vivo models of disease
(Chaum, 2002). The catalytic activity of ribozymes engineered to target and cleave specific
mRNA mutations has been demonstrated in animal models in vitro. Ribozyme therapy is Comment [N5]: This wording is imprecise and
confusing, suggesting that the student has not
effective in animal models. Despite the efficacy of ribozymes to eliminate mutant transcripts, understood a key distinction in experimental
work. The mention of animal models implies in
ribozymes require repeat treatments, sustained delivery devices, or transduction by integrating vivo experiments, not in vitro experiments.
vectors to achieve sustained transgene expression (Chaum, 2002). Comment [N6]: No evidence is provided for
this statement.
Antisense gene therapy is based upon the use of synthetic, short DNA sequences
(oligodeoxynucleotides, ODN) that are designed to be complementary to a targeted mRNA
molecule (Chaum, 2002) The ODN is capable of forming a stable DNA-RNA heteroduplex with
the mRNA and, thus, prevent translation of the protein from the transcript (Chaum, 2002). The
mRNA of the heteroduplex is enzymatically destroyed by RNase H, but the ODN is not, so that
the ODN is free to bind to other identical mRNA transcripts, and function catalytically, in a
manner similar to ribozymes, to suppress translation of a specific gene transcript (Chaum, 2002). Comment [N7]: This paragraph lacks an
introductory or concluding sentence indicating
its purpose and relationship to the overall topic
Cell culture is a technique used to create cell lines that can replicate themselves for of the mini-review. It summarizes information,
but does not seem to have a clear purpose. In
hundreds of generations (Shimidzu, 2002). Such cell lines are cultured in a specific medium and particular, it neglects to state how the technique
are used in many instances to replace whole animals in medical experiments (Shimidzu, 2002). being discussed compares with transgenics.

Cell cultures are cheap to use and relatively easy to maintain. The main problem in using cell
cultures, however, has been the unpredictable nature of new cell lines (Shimidzu, 2002). Most
cell lines now in research use have been derived from cancer cells because normal cells die out
after about fifty generations (Shimidzu, 2002). The reason why cells die or at least become
defective after a certain number of generations is not well known (Shimidzu, 2002). Telomere
length is thought to play a significant role in cell death. Cancer cells, through an unknown
mechanism, can maintain telomere length through generation (Shimidzu, 2002). In addition to Comment [N8]: The connections between
these sentences are hard to follow. It is also not
the unpredictable nature of creating new cell lines, it is also difficult to culture cell lines that are clear how they relate to the topic of the mini-
review. 
free of unwanted cells or organisms (Shimidzu, 2002). Almost always some sort of antibiotic
agent has to be used which makes creating cell lines less reliable for use in research. Moreover,
some kinds of contaminations such as contamination by mycoplasms are not always readily
detectable (Shimidzu, 2002).

Episomal expression is one the most promising alternatives to transgenic technology

(Isner, 2002). Basically, episomal expression consists of inducing the expression of novel
proteins inside the cell by transient expression of DNA inside the cell (Isner, 2002). This method
relies on a variety of vectors to transfer the desired DNA sequence to the interior of the cell.
Viral vectors are used when short expression times are required and non-viral vectors are used
when longer expression times are needed (Isner, 2002). Adenoviruses, however, are not used
because using an adenovirus mean that the DNA will be incorporated into the cell’s genome
(which by definition would make the cell transgenic). Two main difficulties faced by the
episomal expression approach are the fact that sometimes introduction of DNA into a cell in
large quantities may kill the cell and that some types of cells are impenetrable to known vectors.
Therefore getting the DNA into them is a challenge on its own (Isner, 2002).

Growth factors are either second messengers or agents that cause release of second
messengers. They trigger cellular processes through manipulating the signal transduction system
of the cell. Growth factors often cause gene expression and subsequent protein production.
Although sometime growth factors are used in research to achieve a certain physiological effect
in a cell or tissue the use of growth factors in hampered by the fact that growth factors tend to
affect a family of cellular receptors. So growth factors would tend to produce a cascade of
unwanted reactions and lead to undesirable side effects. Gene silencing by ribozymes and Comment [N9]: This passage is not directly
related to the topic of transgenics and
antisense oligonucleotides, cell culture, episomal expression vectors, and growth factor treatment alternative techniques, and should therefore be
omitted. 
are alternatives to the transgenic technology for use in medical research, however, each of these
methods posses its own unique difficulties. Comment [N10]: This conclusion is very
weak: it merely restates the topic. It neglects to
give an opinion on whether transgenics can be
replaced with alternative techniques.
References:

Chaum, E., & Hatton, M. (2002). Gene Therapy for Genetic and Acquired Retinal
Diseases. Survey of Ophthalmology. 47, 449-469(2002)

Isner, J. Myocardial gene therapy. Nature 415, 234 - 239 (2002)

Shimidzu, Y. Ishida, T. Spontaneous establishment of a novel Japanese macaque cell line with
epithelial cell phenotypes. In Vitro Cell. Dev. Biol. —Animal 38:311-313 (2002) Comment [N11]: In the ICMJE citation-
sequence system, this entry would appear as
follows: ''1. Chaum E, Hatton M. Gene Therapy
  for Genetic and Acquired Retinal Diseases.
Survey of Ophthalmology 2002;47:449-469.'' It
would appear first in the Reference List
GENERAL COMMENTS because it was the first source mentioned in the
text of the paper. Note the streamlined
punctuation, tight spacing, and lack of italics.
Strengths: The description of alternative methodologies is generally clear. Grammar is correct The date comes after the title of the journal.
 
and style and tone are appropriate.

Needs improvement: This paper lacks focus. It does not define transgenics or explain why it
may be necessary to look for alternative techniques. It summarizes information about research
techniques, but makes few connections among ideas. The paper gives only one citation per
technique, suggesting that inadequate reading may explain the lack of insight displayed. The
reader is left with the impression that the material has been regurgitated from other sources
without a full understanding. There are several errors in the use of technical terms and in
referencing format.