A method for multiplex gene synthesis employing error correction based on expression.

Our ability to engineer organisms with new biosynthetic pathways and genetic circuits is limited by the availability of protein characterization data and the cost of synthetic DNA. With new tools for reading and writing DNA, there are opportunities for scalable assays that more efficiently and cost effectively mine for biochemical protein characteristics. To that end, we have developed the Multiplex Library Synthesis and Expression Correction (MuLSEC) method for rapid assembly, error correction, and expression characterization of many genes as a pooled library. This methodology enables gene synthesis from microarray-synthesized oligonucleotide pools with a one-pot technique, eliminating the need for robotic liquid handling. Post assembly, the gene library is subjected to an ampicillin based quality control selection, which serves as both an error correction step and a selection for proteins that are properly expressed and folded in E. coli. Next generation sequencing of post selection DNA enables quantitative analysis of gene expression characteristics. We demonstrate the feasibility of this approach by building and testing over 90 genes for empirical evidence of soluble expression. This technique reduces the problem of part characterization to multiplex oligonucleotide synthesis and deep sequencing, two technologies under extensive development with projected cost reduction.

How the Kennel Club is tackling inherited disorders in the United Kingdom.

Health screening of potential canine breeding stock can provide invaluable information to allow breeders to select against inherited diseases in their breeding programmes. This review details the screening programmes that are currently available to UK dog breeders and evaluates their impact as selective tools for dog breeders.

Population structure and inbreeding from pedigree analysis of purebred dogs.

Dogs are of increasing interest as models for human diseases, and many canine population-association studies are beginning to emerge. The choice of breeds for such studies should be informed by a knowledge of factors such as inbreeding, genetic diversity, and population structure, which are likely to depend on breed-specific selective breeding patterns. To address the lack of such studies we have exploited one of the world's most extensive resources for canine population-genetics studies: the United Kingdom (UK) Kennel Club registration database. We chose 10 representative breeds and analyzed their pedigrees since electronic records were established around 1970, corresponding to about eight generations before present. We find extremely inbred dogs in each breed except the greyhound and estimate an inbreeding effective population size between 40 and 80 for all but 2 breeds. For all but 3 breeds, >90% of unique genetic variants are lost over six generations, indicating a dramatic effect of breeding patterns on genetic diversity. We introduce a novel index Psi for measuring population structure directly from the pedigree and use it to identify subpopulations in several breeds. As well as informing the design of canine population genetics studies, our results have implications for breeding practices to enhance canine welfare.

A testicular influence on restraint-induced activation of medial parvocellular neurons in the paraventricular nucleus in the male rat.

To gauge the strength by which the testes influence stress-induced activation of neurosecretory neurons in the paraventricular nucleus, we studied within medial parvocellular neurons the effects of gonadectomy on restraint-induced Fos-immunoreactivity and on CRH and arginine vasopressin (AVP) heteronuclear (hn) RNA expression levels. Relative to intact male rats (sham-gonadectomized), gonadectomized rats showed a significantly greater number of medial parvocellular neurons recruited to express Fos protein evident at 0.5 h and from 1-4 h after the onset of 30-min restraint exposure. Restraint provoked a transient increase in hnCRH levels that was maximal at the end of restraint and this was significant only in gonadectomized rats. Both intact and gonadectomized rats displayed an increase in AVP hnRNA expression levels in response to restraint exposure; however, it was significantly greater in gonadectomized rats. All of these responses were accompanied by a higher corticosterone response in gonadectomized compared with intact rats and negatively correlated with plasma testosterone concentrations, with the exception of stress-induced CRH transcription. These findings indicate an inhibitory role for testosterone on stress-induced indexes of synaptic (Fos) and transcriptional (AVP hnRNA) activation among hypophysiotropic paraventricular neurons and provide meaningful end points with which to pursue how and where androgens operate on stress-related input to the paraventricular nucleus motor neurons.