376 CHAPTER 15mechanisms also affect the expression of genes and their products. One is DNA
methylation. In some organisms, such as vertebrates and plants, transcription of a
gene is repressed by methylation of certain cytosines that are followed by guanine
residues (CpG dinucleotides). The methylated state may be maintained in newly
synthesized DNA during cell division by a specific enzyme. Gene expression can
also be affected at the level of translation of mRNA to protein. For example, micro-
RNAs, together with proteins, bind to the 3′ untranslated region (UTR) of RNA
messages and prevent translation. And various posttranslational processes can
affect the activity of proteins, such as binding a signaling molecule. Recall also that
the various exons of a eukaryotic gene are often differentially spliced into many
isoforms that have different functions and may be expressed in different cellsWe briefly describe here some methods commonly used to
study genes that underlie development, and their expres-
sion as mRNA transcripts. More detailed descriptions can be
found at devbio.com; search for topic numbers 2.3 and 2.4.
This is the website of a leading textbook, Developmental
Biology by Scott F. Gilbert and Michael J. F. Barresi [19].
A gene from one organism can be inserted into the
genome of another (often a different species), creating a
transgenic organism. This can be accomplished by micro-
injection, usually into a fertilized egg; by electroporation
(using a high voltage pulse to “push” the DNA into the egg);
or by attaching the gene to a transposable element or to a
retrovirus vector, which inserts the gene into the host’s ge-
nome. Gene function is also studied by gene knockout (or
knockdown), which uses RNA interference (RNAi) to prevent
transcription. It is also possible to replace the normal gene
with a nonfunctional mutated sequence, using CRISPR-Cas
targeted mutagenesis (FIGURE 15.A1A).
Several methods are used to visualize and measure
transcription of specific genes in various cell populations
at different times in development. In in situ hybridization, a
chemical process stabilizes mRNA molecules in the cells in
which they are produced. Then a species-specific, single-
stranded RNA or DNA probe corresponding to the gene
of interest is applied to the specimen, where the probe
hybridizes by base pairing with the mRNA of interest. The
probe is chemically modified so that it can be detected by a
staining procedure or is labeled with a radioisotope so that
it can be detected by autoradiography.
Another approach is to use RNA-seq and related methods
to measure the entire transcriptome—the expression of all the
genes at once. Levels of protein expression can be analyzedusing mass spectrometry or antibodies. (The mRNA and pro-
tein expression patterns of a given gene may not be identical
due to translational regulation.) Antibodies are produced
by injecting a mammal (e.g., a rat) with the protein of inter-
est (the antigen). The animal produces antibodies (immu-
noglobulin molecules) that bind specifically to that protein.
Tissue specimens are prepared in a similar way as for in situ
hybridization and are incubated with the primary antibody.
A secondary antibody, an immunoglobulin that specifically
binds to the primary antibody, is then applied to the speci-
men. The secondary antibody is modified so that it can be
detected either by an enzymatic reaction that produces a
colored product or by fluorescence (FIGURE 15.A1B).
Finally, the transcription patterns of cis-regulated genes
can be studied using reporter constructs inserted into
cultured cells or transgenic individuals. Reporter constructs
consist of the regulatory DNA of interest, spliced upstream
of a reporter gene that encodes a protein whose expres-
sion can be easily visualized under the microscope. One
such protein is β-galactosidase, a bacterial enzyme that
processes a particular sugar into a blue product. Another is
a protein from jellyfishes (green fluorescent protein, GFP)
that fluoresces bright green. Because reporter construct
analysis requires the use of gene transfer technology, it can
be undertaken only in certain well-studied model species,
such as Drosophila, Caenorhabditis elegans, Arabidopsis,
and mice. FIGURE 15.A1C shows the nematode C. briggsae
expressing a GFP reporter construct containing cis-regula-
tory DNA from the myo-2 gene, which directs the reporter
gene’s expression in the pharynx.
In genetic model species, the integration of genomic with
genetic, developmental, and functional data provides aBOX 15A
Some Methods in Developmental Genetics
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