One of the most powerful
experimental techniques in current biomedical research is the ability to alter
the genes that ultimately control all cellular processes. Individual genes can
be removed from the DNA of a variety of organisms from bacteria and yeast to
fruit flies, fish and mice. The “knock-out” organisms created by this
process often exhibit characteristics that reveal important information about
the function of the missing gene’s product. Furthermore, the majority of
genetic diseases are the result of similar, though naturally-occurring,
perturbations to human genes and “knock–out” mouse models for such
diseases are often of great utility in their study.
Unfortunately, gene alteration in
mice is a complex process involving considerable time, effort and expense.
Also, perturbing some genes may create a condition that is not compatible with
development, greatly reducing the utility of the technique. There is another
way to perturb gene function and that is to interfere with the process of
expression through which the cell actually uses the gene. Over the years, a
variety of methodologies have been proposed for interfering with gene
expression but, until recent advances, none of these have been particularly
effective. A new technique known as double-stranded RNA interference (RNAi) is
proving to be a revolutionary method for interfering in a very specific and
powerful way with the expression of target genes. RNAi has not only shown
great promise as a basic research technique, it also may have great potential
use in clinical protocols such as those directed against viruses like
HIV.
RNAi seems to be an ideal technique
particularly for looking at issues during early development. Using RNAi, the
effect of individual genes can be dissected during the early embryonic
divisions. Since this critical developmental period is of primary concern to
protocols involved with human assisted reproduction, RNAi is a perfect
technique for research in this area. The underlying causes of human
infertility may involve deficits in gene function during sperm and egg
development, fertilization, chromosome movement, and the first few divisions
of the early embryo. Using RNAi, genes thought to play a role in these
processes can be individually suppressed, hopefully revealing a specific
effect. One way to use RNAi would involve perturbing the function of
reproductive-critical genes in early mouse embryos to develop animal models
for human reproductive dysfunctions. Also, RNAi could be applied in discarded
abnormal human embryos to allow for direct analysis of gene function.
RNAi works through the introduction
of many thousands of copies of an RNA “construct” that matches the
sequence of the target gene. Such a construct can be made in a variety of ways
and commercial kits for RNAi construct protocols are available. Multiple
copies of the construct are required since when a gene is “expressed”
multiple copies of the gene message are produced by the cell and it is these
messages that the RNAi construct associates with and inactivates. The cellular
mechanism of RNAi is not fully understood but essentially the RNAi construct
binds to its target native gene message and this binding brings about the
unique destruction of the message by the cell. It is thought that the RNAi
mechanism may be part of an ancestral “cellular immunity” system through
which the cell deals with foreign genetic material, such as viruses. In fact,
clinical protocols using RNAi constructs are currently in development to
address a variety of viral infection issues.
In practice, a solution containing
the RNAi constructs is injected into the cell of interest such as a mouse egg
or 1-cell fertilized egg. This is a relatively simple matter using a fine
glass needle and basic cell micromanipulation equipment – such as is used
for human ICSI. Alternatively, it may be possible to simply place the cell in
a solution of the RNAi construct along with chemical agents that facilitate
entry into the cell membrane. This would be important for treating
multiple-celled structures such as a cleaving embryo. The introduced construct
then quickly binds to and destroys its target message and an effect can be
observed within a matter of hours or days.
Our laboratory has used RNAi to
disturb the function of two genes in early mouse embryos. One of these genes,
Cadherin, is involved with the connections that hold the multi-celled embryo
together. The cadherin protein which is found on the surface of embryonic
cells and mediates cell-cell attachment.
Fig 1. Cadherin RNAi mouse embryos
with control blastocysts on the right.
Cadherin’s role is particularly
important as the embryo develops into a hollow ball – the blastocyst. The
blastocyst contains a central cavity filled with fluid and cell-cell
attachment via cadherin is critical to maintaining the integrity of this
cavity. Mouse embryos injected at
the one cell stage with a cadherin RNAi construct exhibit normal development
up until the blastocyst stage at which point they essentially “fall apart”
and fail to form an intact blastocyst cavity. This is illustrated in figure 1
with “control” embryos injected with a non-functional RNA construct
exhibiting normal blastocyst development.
The cadherin result had already been established through prior
experiments and was pursued as a test of the RNAi system.
A second novel gene that we have
targeted is survivin. This gene plays a role in regulating the cyclic process
through which a single cell divides into two “daughter” cells, four cells
and so forth. Such divisions during early development, particularly in the
human, are often prone to errors leading to the production of abnormal
daughter cells and their subsequent dysfunction and cell death. We chose the
survivin gene as a potential target involved in pathological division issues
since it seems to play a role in determining if division has proceeded
properly. In fact, when survivin was perturbed using RNAi, mouse embryos
exhibited a variety of division abnormalities leading to the type of cellular
fragmentation often observed in human embryos (Fig. 2). Mouse embryos do not
normally exhibit fragmentation type behavior unless treated with toxic
compounds so this result creates an important animal model for studying
fragmentation. Ongoing experiments have identified specific abnormalities in
cellular components that may be involved in the abnormal cell divisions that
create the fragmented structures. Continued study of these embryos will
hopefully reveal further insights into the basis of such abnormalities.
Fig. 2 Survivin RNAi mouse embryo
and fragmented human embryo
RNAi shows great promise for the
future in analyzing the role of specific genes important to human
reproduction. Such experiments are the perfect partner for ongoing studies to
identify such critical genes from expression studies in human eggs and early
embryos. Together, these experiments will, for the first time, give us a clear
picture of the underlying mechanisms of early human development. With such
knowledge in hand, clinical protocols can be optimized and even directed
towards patient-specific requirements. Also, such knowledge will also allow us
to identify the best quality embryos for replacement, ensuring the most
positive outcome possible in every ART cycle.
