Animal Biotechnology and Modeling
233
technique, Northern blot hybridization,
confrms not only the presence but also
the size oF the transcript oF interest. The
Northern blotting technique consists oF
electrophoresis into
an
agarose
gel
to
separate the RNA molecules on the basis
oF size, transFer (blotting) oF the RNA
to a nylon or nitrocellulose membrane,
and detection oF the mRNA oF interest by
hybridizing it to a specifc, radiolabeled
probe, Followed by autoradiography.
Additional techniques exist For deter-
mining the presence oF relative levels oF
mRNA transcripts From transgenic ani-
mals. In the nuclease protection assay, a
radiolabeled probe is allowed to hybridize
to the RNA in solution, Followed by nucle-
ase digestion oF unhybridized RNA. The
sample is then resolved on a polyacry-
lamide gel, Followed by autoradiography to
detect the probe. This technique is very
useFul in determining the steady state lev-
els oF RNA in a given tissue and oFFers an
inherently high signal-to-noise ratio. In the
reverse transcription-PCR (RT-PCR) assay,
the RNA oF interest is transcribed into a
cDNA molecule by means oF a specifc
primer and the enzyme reverse transcrip-
tase. A second primer is added, and a
standard PCR amplifcation is perFormed.
The
advantage
oF
RT-PCR
lies
in
its extreme sensitivity – theoretically, one
mRNA molecule can be amplifed to a
quantity suFfcient For visualization on an
agarose gel aFter ethidium bromide stain-
ing. The drawbacks include the possibility
oF genomic DNA contamination, in ad-
dition to the concerns associated with
standard PCR reactions (e.g. False posi-
tives, mispriming, contamination, etc.).
In the
in situ
hybridization technique,
a labeled probe is hybridized to a target
mRNA transcript in sections oF tissue to
permit the identifcation oF individual cells
containing the transcript. This technique is
particularly useFul in identiFying subsets oF
positive cells within a given tissue in which
the relative levels oF specifc transcript For
the tissue as a whole might be too low to
be detectable by other means.
4.3
Protein Expression
In addition to the Foregoing assays For tran-
scription oF the transgene, various other
techniques to determine the translation oF
the transcript into a protein product are
oFten employed. Most oF these depend on
the availability oF specifc antibodies to the
protein oF interest. The immunoblotting
technique, in which proteins are resolved
on a polyacrylamide gel, transFerred to a
membrane, and detected by means oF a
labeled antibody, is useFul in veriFying the
appropriate molecular weight oF the pro-
tein product oF interest. Although rarely
used, the radioimmunoprecipitation assay
can also serve in the determination oF the
size oF the protein product and to allow
For various other manipulations, including
peptide mapping and isoelectric Focusing,
to veriFy proper modifcation oF the protein.
To identiFy the cells within a tissue that
contain the protein product oF the trans-
gene, immunohistochemical ‘‘staining’’ oF
tissue sections with a labeled antibody is
oFten employed.
Some experiments involving transgenic
animal production Focus on measuring the
influence oF various cis-regulating DNA
sequences; the transcript and ultimate
protein product are For the most part ir-
relevant. In these cases, ‘‘reporter genes’’
are oFten employed because they can sim-
pliFy determination oF expression levels by
producing a transcript or protein that is
easily and unequivocally detectable. Par-
ticularly useFul in these applications are
reporter genes that code For novel enzymes
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