Improving reproductive cloning

Remember when Dolly the sheep was cloned in 1996?  That was the first cloned mammal and everyone freaked out thinking we would be cloning all our pets and even humans within a few years.  Well, nearly 20 years have passed since then and reproductive cloning is still a very difficult and inefficient procedure.  Most cloning has a 1-5% success rate.  Why is that?  Before we can answer that, we need to understand the procedure for reproductive cloning.

Somatic cell nuclear transfer

Our bodies are made up lots of different types of cells – neurons, skeletal muscle, intestinal cells, immune cells, etc.  Despite the different functions and structures of these cells, all of the cells in one organism have the same genome, the same set of genes.  What makes cells unique is that they express different genes at different times, so different proteins are made.  What this means is that the blueprint (DNA) for making a new organism is right there in every cell in your body.

The normal way of making an embryo is by taking half of the genome from a male (in sperm) and half from a female (in the egg) and combining them during fertilization.  In reproductive cloning, you already have a whole genome from any adult cell.  That nucleus from the adult can be inserted into an oocyte (or egg) that has had its DNA removed (“enucleated”).  The egg is necessary because it has lots of nutrients and signals in it that are important for the first few cell divisions during early development.

This process is shown in the diagram below and is called somatic cell nuclear transfer.  In the example, the adult genome is coming from a fibroblast cell and is transferred into an enucleated oocyte.  This is a way to get stem cells (ntES), which can be used for therapeutic purposes, like making more neurons that can be transplanted into someone with Parkinson’s.  Or you could let the cloned cells grow up into an embryo and then into a cloned organism.

Somatic nuclear cell transfer (from Stembooks.org)

Although it is possible to make cloned organisms using adult donor genomes, the efficiency is much higher when using genomes from embryos.  What happens to the adult genome that prevents it from directing the formation of a new organism?  This problem is addressed in a recent paper by Matoba et al., published in Cell.

Epigenetic changes

Although adult cells should have the same DNA sequences as their embryonic precursors, the genome can be organized differently, which can affect which genes are expressed.  DNA wraps around histone proteins as a way to organize the long DNA chains.  Histones can be modified in such a way that the DNA will wrap around more tightly or more loosely.  For example, if a particular amino acid in histone 3 is trimethylated (three CH3 groups are added), then that makes the DNA pack up closer together, so it is really hard to express those genes.  There are genes that may need to be expressed early on in development, so their histones will be modified to allow for loose packing, but then after they are expressed, they’ll get packed away, so they take up less space.  These kinds of modifications that affect gene expression are called epigenetics.

As an analogy, imagine you have had a child, so you have baby clothes, a crib, car seat and toys in your house.  Once that child grows up, you take all those baby things down to the basement.  You still have them, but you will probably never need to use them again, so you can pack them all up and store them so they are out of the way.  It may be hard to access them again, but they are still there.  So a gene that has been packed away into condensed chromatin is still present in a cell, but it is no longer giving instructions for making proteins, unless something comes along and unpacks it.

You can see now the problem with somatic cell nuclear cloning.  The adult cell already has some DNA packed away, so when the genome is put into an oocyte, it may be impossible to express the genes necessary to direct normal development.  In the paper by Matoba et al., they did indeed find that there are more trimethyl modifications (called H3K9me3) in mouse embryos derived from nuclear transfer than embryos from in vitro fertilization (using a sperm and egg).  These regions were associated with decreased gene expression and compact DNA.

Improving efficiency

Now we know one of the problems, but what can researchers do to improve the efficiency of reproductive cloning?  Somehow they need to decrease H3K9me3 modifications in the donor genome.  They do this two ways:

(1) There are too many methylated histones, so the authors injected an enzyme that removes methyl groups into the one-cell embryos.  The embryos expressed more genes and survived throughout development.  70% of these cloned embryos implanted into a surrogate mouse uterus and 8% survived to adulthood.  Those numbers are higher than before, but still not perfect.

(2) They also tried decreasing expression of the enzymes that put on the methyl groups.  This also improved development, so 50% of the embryos made it to later stages of development, but they did not see how many survived to adulthood.

There is an epigenetic barrier for nuclear transfer from adult cells in mouse oocytes.  Presumably a similar problem is preventing cloning in other organisms as well.  It makes sense that an adult cell would have a different pattern of epigenetic modifications than an embryonic genome.  The authors were able to improve cloning efficiency by decreasing the H3K9me3 modification, but there are probably other histone modifications that are also different in adults.  There is still a long way to go before cloning is a reliable procedure, but at least now we have some explanation of why it is so difficult.