Over twenty years ago, somatic cell nuclear transfer (SCNT) let scientists successfully clone the first mammal—Dolly the sheep. Despite advances since then, the efficiency of this process remains low. In the July issue of G3, Srirattana and St. John report a method to deplete cattle donor cells of mitochondrial DNA and efficiently generate blastocysts from these depleted donors.

In SCNT, a nucleus taken from a mature, differentiated somatic donor cell is placed into an oocyte whose nucleus has been removed. Together, this newly created cell goes on to form an embryo whose nuclear genome is identical to the donor cell’s. The low efficiency of SCNT is thought to be partially due to incomplete epigenetic reprogramming of the new embryo. In fact, treatment of embryos with the histone deacetylase inhibitor trichostatin A (TSA) is widely used to enhance SCNT in mouse.

Nuclear DNA isn’t the only genetic information in the cell, though. Mitochondria house a distinct genome that is maternally inherited, and mitochondrial DNA (mtDNA) from donor cells can be randomly transmitted from the donor cell to the embryo. The mixing of mtDNA from the two initial cells complicates the process of SCNT.

Normally, an organism inherits its mtDNA from its mother, but SCNT embryos have two different sets of mtDNA—donor cell and oocyte—to deal with, and the interplay between the two can cause genomic instability, aneuploidy, poor embryo quality and implantation rates, and metabolic defects.

To combat this problem, Srirattana and St. John depleted the mtDNA from cattle donor cells by using 2’,3’-dideoxycytidine (ddC), which inhibits the mtDNA-specific DNA polymerase gamma but doesn’t affect nuclear DNA. They compared embryos made from mtDNA-depleted cells to nondepleted cells, and they looked at the effect of TSA treatment on both depleted and nondepleted cells.

They found that mtDNA-depleted donor cells can generate viable blastocysts that contain only oocyte mtDNA—as is the case in blastocysts formed by sperm fertilization. Donors depleted of mtDNA had a lower rate of blastocyst generation than nondepleted cells, but the use of TSA brought the blastocyst rate back to that of nondepleted donors. Srirattana and St. John also showed that gene expression differed between blastocysts created from depleted and nondepleted cells; they found that differentially expressed genes were largely involved in embryonic development, suggesting the use of mtDNA-depleted donor cells can alter development of the cloned embryos.

This work demonstrates that depleting mtDNA from donor cells is an effective tool for creating SCNT embryos with mitochondrial genomes inherited only from the oocyte. Such embryos will hopefully avoid some of the problems created by mixing incompatible mitochondrial genomes and may one day make SCNT easier and more efficient for cattle scientists and others who rely on this cloning technique.

CITATION:

Manipulating the Mitochondrial Genome To Enhance Cattle Embryo Development
Kanokwan Srirattana, Justin C. St. John
G3: Genes, Genomes, Genetics July 2017 7: 2065-2080;
https://doi.org/10.1534/g3.117.042655
http://www.g3journal.org/content/7/7/2065

Thanks to more than a hundred years of working with Drosophila melanogaster, geneticists have many powerful tools for precisely manipulating its genes. It has also become a model system for studying speciation and molecular evolution together with the other members of the melanogaster species group: D. simulans, D. mauritiana, D. yakuba, and D. santomea. However, evolutionary genetic studies have been hampered by an inability to make transgenic lines within the less well-studied species. In the April issue of G3, Stern et al. present a panel of new transgenic strains of these species designed to make fine mapping and functional genetic studies possible. These resources provide new opportunities for unraveling the genetic mechanisms of speciation and evolution.

Transposable elements are selfish genetic elements that insert themselves into new genome locations. This natural machinery has been adapted by scientists to introduce carefully designed DNA constructs into genomes. Stern and colleagues used the piggyBac transposon system to insert a fluorescent reporter gene into hundreds of random locations in the genomes of five different Drosophila species. They mapped the location of each insertion and identified the unique insertions that could be maintained in homozygotes, resulting in 184 D. simulans lines, 122 D. mauritiana lines, 104 D. yakuba lines, and 64 D. santomea lines. Each line has an insertion in a unique genomic location coupled to a fluorescent reporter gene whose expression can be easily detected in the eyes with a microscope. The huge collection of unique locations will make fine mapping in these species much easier.

Coupled to many of the insertions is a landing site that can be used to add new insertions via plasmids carrying a suitable targeting sequence. The authors tested the integration efficiency at landing sites in unique genomic locations and identified those with high efficiency that could be maintained as homozygotes. They also tested the effects of insertion location on expression by transforming a fluorescent reporter gene linked to an enhancer of even-skipped, a developmental gene in D. melanogaster with well characterized expression patterns. In four of the five species tested, at least one strain showed the expected patterns of expression, suggesting that any gene of interest transformed into these sites will not be subject to ectopic expression from location effects. Additionally, they used CRISPR/Cas9 gene editing to knock out expression of the fluorescent eye reporter in a number of strains. Since strains still have the landing site in known locations, they can be used to examine expression of genes of interest in the eye without interference from the reporter.

Noni fruit is toxic to most other fruit flies, but <i>D. sechellia</i> loves it. Photo by Carmen via Flickr.

The fly species of D. melanogaster species group are notable not just for their close relationship with the famous D. melanogaster, but they also make excellent evolutionary models in their own right. D. melanogaster and D. simulans are cosmopolitan species that live wherever humans do, but the other members of the group are found only on islands off the coast of Africa. D. sechellia is a member of the group that feeds primarily on a fruit toxic to most other animals. Despite the adaptive differences that come with these divergent lifestyles, many of these species can still hybridize, making laboratory studies aimed at dissecting these differences possible. The transgenic lines presented in this paper will make studies on speciation and adaptation in these lineages more powerful and accessible than ever before.

Stern, D. L., Crocker, J., Ding, Y., Frankel, N., Kappes, G., Kim, E., Kuzmickas, R., Lemire, A., Mast, J.D. & Picard, S. (2017). Genetic and Transgenic Reagents for Drosophila simulans, D. mauritiana, D. yakuba, D. santomea, and D. virilis. G3: Genes, Genomes, Genetics, 7(4), 1339-1347.

http://www.g3journal.org/content/7/4/1339

When geneticist Rob Unckless took his son to Lego Club at the local library, he was not expecting to start a new collaboration. The result is the striking piece of science-inspired art that graces the cover of the February issue of GENETICS.

Created by artist Kent Smith, “Attack of the 50 Foot Mosquito” was inspired by a paper by Unckless and his colleagues. The study examines a more subtle threat than a rampaging giant: the potential evolution of resistance to gene drives. Gene drives are a burgeoning new technology that use CRISPR-Cas9 genome editing to alter the genomes of an entire population. Cas9 is an enzyme that, when introduced into a cell, makes cuts in DNA that are then repaired through the endogenous homology-directed repair pathway. This repair process can be co-opted to change the final sequence; if a DNA sequence is introduced that carries the desired edit along with some homology to the cut DNA, it can serve as the template for repair. To turn this editing process into a gene drive, Cas9 and the guide RNA are also inserted permanently into the genome, so theoretically every heterozygote will automatically undergo this process. After the first transformation, the gene drive is self-propagating. This technology has been suggested as a way to control populations of disease vectors, like mosquitoes.

Though this technology is exciting and new, selfish genetic elements are definitely not. There are a multitude of natural examples of elements that manipulate their way into more than half of an individual’s offspring. And notably, many of these natural driving elements are associated with genetic suppressors that prevent their selfish activity and restore equal transmission of both alleles. In their paper, Unckless and colleagues discuss potential mechanisms of resistance to gene drive, including new mutations and accidental by-products of the repair process involved in gene drive. They use mathematical modeling to show the probability that resistance spreads through the population is very much dependent on the frequency at which resistant alleles arise in the first place. This study shows much care and study is needed to more effectively use gene drives in the wild.

This stunning cover was born when Unckless had just moved to Lawrence to begin an assistant professor position at the University of Kansas. He took his son to the Lego Club at the library; Kent Smith, a local artist who teaches in the School of Architecture and Design at KU and  specializes in science fiction-inspired artwork, was also there with his son. “I struck up a conversation with Kent,” says Unckless, “We talked about what I do and what he does, and decided it would be fun to collaborate on something.” When the manuscript was accepted by GENETICS, they decided to do a piece to submit as a potential cover.

“I loved being able to provide a fun visual for all of this amazing big brain science!” says Smith. “One of my favorite things about design and illustration is getting to research and learn about new subjects for each piece.” Unckless and Smith discussed what the paper showed about gene drives, and then Smith came up with a way to illustrate the evolution of resistance metaphorically. “Nature’s response to the gene drives comes in the form of a giant monster mosquito rampaging through a city,” he explains. The piece is a reference to the iconic poster for the 1958 science fiction movie Attack of the 50 Foot Woman. Unckless saw rough sketches and gave suggestions about things like mosquito anatomy and the mosquito-borne Zika, dengue, chikungunya, and yellow fever viruses swirling in the clouds of smoke and destruction. A tiny Andy Clark can also be seen fleeing destruction on his bike.

“I was lucky to have such a rich subject matter,” says Smith. “Collaboration and dialogue with Rob was very inspirational and allowed for some great brainstorming.”

The final product of this meeting between art and science is a tongue-in-cheek commentary on the potential perils of gene drive gone awry. Though rampaging giants are unlikely to result, scientists should carefully consider the consequences of this potentially powerful technology.

Unckless, R. L., Clark, A. G., & Messer, P. W. (2017). Evolution of resistance against CRISPR/Cas9 gene drive. GENETICS. 205(2):827-841. DOI: 10.1534/genetics.116.197285

http://www.genetics.org/content/205/2/827

More of Kent Smith’s artwork can be found here:

www.smittytown.com

https://www.facebook.com/KentSmithIllustration/

https://www.instagram.com/smittytownart/

https://society6.com/smittytown