Re: How is the Barr Body used in cloning animals?

Thanks for an interesting question.

First of all, let's discuss what a Barr body is. 

In animals that have an X-Y sex determination system (like mammals), males 
are XY and females are XX. Mammals have two copies of all of their 
chromosomes, one from each parent, except for males, which have a single 
copy of the X and a single copy of the Y (the Y chromosome has a very small 
number of genes in common with the X and a few genes of its own, but is very 
gene-poor). Embryos with abnormalities of chromosome number occur 
occasionally as a consequence of errors of meiosis. Embryos that have only a 
single copy of one of the chromosomes (except the X) are inviable. Embryos 
that have three copies of one of the chromosomes are usually inviable as 
well. One exception to this rule is that humans with an extra copy of 
chromosome 21 are viable, but have Down syndrome. Human chromosome 21 is 
very small and carries relatively few genes.

Abnormalities of chromosome number cause problems due to an imbalance of 
gene dosage. Many genes work together in any particular metabolic or 
developmental pathway. Increasing or decreasing the number of copies of a 
gene usually increases or decreases the level of expression of that gene. If 
only one or a few genes are involved, the effects are usually minor, but 
when hundreds of genes are present in only one copy or in three copies 
instead of two copies, many metabolic or developmental processes are 
affected, and this is often fatal to a developing embryo.

Study of the human genome has revealed that the X chromosome has about three 
times as many genes as chromosome 21, yet monosomy for the X chromosome (the 
state of having only one copy, normal for males) does not produce 
developmental abnormalities. Having an extra copy of the X chromosome (as 
females do, from the standpoint of males) does not produce developmental 
abnormalities. The reason is that the genes on the X chromosome are 
expressed at a level that produces the right balance of gene products with 
autosomal genes when there is a single X chromosome. Females avoid gene 
imbalance because in each of their cells, one of the X chromosomes is 
inactivated by being condensed into a highly compact state called a Barr 
body. Almost none of the genes on the inactive X chromosome can be 
transcribed (expressed).

Because males do not have a Barr body, and male mice, sheep, pigs, goats, 
and cattle have all been cloned, it follows that cells do not require a Barr 
body in order to be used for cloning. This is a trivial answer to your 
question.

Let's talk exclusively about female cells for most of the rest of this 
answer.

When a female embryo is created by fertilization, it has a maternally-
derived X chromosome (from its mother) and a paternally-derived X chromosome 
(from its father). Early in development, both X chromosomes are active. This 
tells you that either X chromosome that could be inherited from the mother 
would have to be active in the developing germ cells. (Germ cells reactivate 
the inactive X in the course of their development.) About the time of 
implantation, when the embryo reaches about 50 - 100 cells, each cell in the 
population will adopt one of two fates: it will either become part of the 
extraembryonic membranes and the placenta, or become part of the embryo 
proper. The cells that will not become part of the embryo are called the 
trophectoderm, while the cells that will be the embryo itself are called the 
inner cell mass. At this stage, each cell decides to inactivate one X 
chromosome. Oddly enough, all of the cells of the trophectoderm will 
inactivate the paternally-derived X chromosome. The cells of the inner cell 
mass will choose an X to inactivate at random. On average, about 50% of the 
cells of the inner cell mass will inactivate the maternally-derived X 
chromosome, while the others will inactivate the paternally-derived X 
chromosome. Once a cell has made this decision, all of the cells that ever 
arise from that cell will have the same X chromosome inactive. Obviously, 
the cells of the trophectoderm can distinguish the maternally-derived X 
chromosome from the paternally-derived X chromosome. We are not sure exactly 
how this works, so we have invented a word for it: "imprinting". The 
paternally-derived X chromosome carries an "imprint", which is maintained 
during cell division. We think that imprinting has something to do with the 
level of methylation of CpG dinucleotides (see references below).

Mature somatic cells from females have an inactive X as a Barr body, while 
the zygotic nucleus of a female embryo has both X chromosomes active. It is 
therefore interesting to think about what happens when we attempt to clone a 
female mammal by nuclear transfer of a somatic nucleus to an enucleated egg. 
Somehow, the inactive X must reactivate. We know this because female clones 
are a mix of cells that have one or the other X chromosome inactivated; they 
are not composed only of cells that have the same X inactive that was 
inactive in the donor nucleus. Oddly enough, the X chromosome selected for 
inactivation in the trophectoderm (remember, normally this is always the 
paternally-derived X chromosome) is always the one that was the inactive X 
chromosome in the donor nucleus. So apparently the cell sees the inactive X 
in the somatic donor nucleus as the equivalent of an imprinted paternal X.

Cloning in mammals has a very low success rate. In embryos derived from 
cumulus cell nuclear transfer, some 1-5% of embryos are apparently normal, 
some 70% are clearly abnormal and cannot survive long after birth if they 
make it that far, and some 25-30% fail early in development. This may be the 
result of the failure of the reprogramming process. Because the success rate 
for male clones is not higher than that for female clones, the reason for 
the high failure rate cannot be a failure of X-chromosome reactivation alone 
(this process does not occur in male clones). Therefore, there are other 
reprogramming events (or epigenetic modifications, to use the fancy term) 
that are difficult to complete successfully in both male and female clones.

You have asked a question in a very complex area that requires a fair amount 
of knowledge about genetics, and the use of many genetic terms. You can see 
definitions of many of the terms that I have used at:

 http://
www.informatics.jax.org/userdocs/glossary.shtml


You can also search PubMed at:

 http://
www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed


I searched using "X inactivation AND cloning" and found some good articles 
and reviews.

For a review of X-inactivation, please see:

1. M. F. Lyon, Curr. Biol. 9: R235 (1999)

Here are two articles on epigenetic modification of the mammalian genome as 
it relates to cloning:

2. Science 293: 1089-93 (2001)
3. Science 293: 1093-1098 (2001)

Here is a cool experiment discussed by both of the reviews above:

4. Science 290: 1578 (2000)

The article above has a short commentary article:

5. Science 290: 1518 (2000)

Thanks again for a great question.

Yours,

Paul Szauter
Mouse Genome Informatics