Mice are the new yeast

(chromosome engineering in mice)

I want to highlight a recent paper that engineered mice in a new way, and might be useful for doing lots of cool biology.

These researchers developed a method for creating targeted fusions of chromosomes, generating new karyotypes.¹ They went about this in a very interesting way:

1. Generate haploid mouse embryonic stem cells from unfertilized eggs. (You might remember me mentioning this before).

2. Generate double-stranded breaks in DNA at the centromere of one chromosome and the telomere of another. By chance, some of the cells will fuse the two targeted chromosomes together.

3. Screen cells (by PCR) for the desired fusions. The researchers isolated cells with fusions of chromosomes 4 and 5, and 1 and 2.² Along the way, they also found some that had a fusion of chromosomes 1 and 17, which was not intended.

4. Delete three genomic regions that posed problems for epigenetic imprinting, to generate an overall sperm-like imprinting pattern.

5. Inject haploid ESCs into oocytes, activate them to begin embryonic development, and implant the embryos into surrogate mice.

The researchers were able to generate live, healthy mouse pups with fusions of chromosomes 4 and 5, reducing the overall number of chromosomes by one. These mice were largely normal, and were able to reproduce, even generating mice homozygous for the engineered chromosome. However, the reproductive success of heterozygotes was somewhat lower than wild-type, due to difficulties in chromosome pairing during meiosis.

Fusions of chromosomes 1 and 2 were also viable, but the mice had some defects, notably including outright sterility. In vitro, fusing the two largest chromosomes (1 and 2) had negative effects on cell division and karyotype stability. The researchers showed that this effect was mainly due to the large size (378 Mb) of the fused chromosome, since truncating it by translocating part of it to chromosome 17 removed these negative effects.

Although similar engineering been done in yeast for quite some time,³ this is the first example of targeted karyotype engineering in a mammal. Haploid stem cells are quite a powerful platform for this. It’s much harder to engineer a desired chromosome fusion or translocation in diploid cells, since with the greater chomosome number there is much less control over how things are ligated back together after cutting.

Overall I am quite excited about this, since it will open up a lot of new opportunities for things like introducing completely new engineered chromosomes.

Also: two interesting papers about artificial mouse embryos came out this summer, if you haven’t already seen them you should definitely take a look here and here.

1. A karyotype is a particular set of chromosomes. For example, my karyotype is 46XY: two copies of each of the the 22 autosomes, one X chromosome, and one Y chromosome.

2. Also note that the researchers needed to sort cells to exclude those that spontaneously diploidized. Growing haploid stem cells isn’t easy!

3. For a very cool example of yeast engineering see here where researchers fused 16 yeast chromosomes together into 2 giant chromosomes! But keep in mind that yeast chromosomes are much smaller (the giant chromosomes were only 6 Mb).