Optimal mating strategies to manage a heterozygous advantage major gene in sheep

Some mutations (or ‘major genes’) have a desirable effect in heterozygous carriers but an undesirable effect in homozygous carriers. When these mutations affect a trait of significant economic importance, their eradication, depending on their effect and frequency, may be counterproductive. This is especially the case of major genes affecting the ovulation rate and thus the prolificacy in meat sheep populations. To manage such situations, a mating design based on the major genotypes of reproducers has to be optimized. Both the effect of the major gene and the cost of genotyping candidates at this locus influence the expected genetic progress and profitability of the breeding plan. The aim of this study was to determine the optimal combination of matings that maximizes profitability at the level of the whole population (nucleus + commercial flocks). A deterministic model was developed and, using sequential quadratic programming methodology, the optimal strategy (optimal combination of matings) that maximized the economic gain achieved by the population across a range of genotype effects and genotyping costs was determined. The optimal strategy was compared with simpler and more practical strategies based on a limited number of parental genotype mating types. Depending on the genotype effect and genotyping costs, the optimal strategy varied, such that either the heterozygous frequency and/or polygenic gain was maximized with a large number of animals genotyped, or when genotyping costs were higher, the optimization led to lower heterozygous frequency and/or polygenic gain with fewer animals genotyped. Comparisons showed that some simpler strategies were close to the optimal strategy. An overlapping model was then derived as an application of the real case of the French Lacaune meat sheep OVI-TEST breeding program. Results showed that a practical strategy based on mating non-carriers to heterozygous carriers was only slightly less effective than the optimal strategy, with a reduction in efficiency from 3% to 8%, depending on the genotyping costs. Based on only two different parental genotype mating types, this strategy would be easy to implement.     

Cryopreservation of transgenic mouse embryos — an eight years’ experience

A total of 70,629 predominantly transgenic mouse embryos prepared from 9,727 pregnant female donors were cryopreserved using a method leading to a high revitalisation rate. Against loss, 125 mutant mouse lines were protected. An average of 7.26 embryos (eight-cell embryos) per pregnant donor was received. To reduce the number of animals required as embryo donors, a special breeding exclusively for cryopreservation was omitted if possible and subsequently the number of animals used for freezing was reduced remarkably. The advantage of this strategy is that (mutant) mouse lines out of current use do not have to be kept in a breeding nucleus.In parallel, this procedure leads to rederivation and improves the export of mice to other facilities. The cryopreservation of these 125 mutant lines keeps the potential to save approximately 20,000 laboratory mice per year to be bred if they were kept in a breeding stock. This is a major contribution to the “3R” requirements developed by Russel and Burch to reduce the number of laboratory animals.     

Total cost estimation for implementing genome-enabled selection in a multi-level swine production system

Background

Determining an animal’s genetic merit using genomic information can improve estimated breeding value (EBV) accuracy; however, the magnitude of the accuracy improvement must be large enough to recover the costs associated with implementing genome-enabled selection. One way to reduce costs is to genotype nucleus herd selection candidates using a low-density chip and to use high-density chip genotyping for animals that are used as parents in the nucleus breeding herd. The objective of this study was to develop a tool to estimate the cost structure associated with incorporating genome-enabled selection into multi-level commercial breeding programs.

Results

For the purpose of this deterministic study, it was assumed that a commercial pig is created from a terminal line sire and a dam that is a cross between two maternal lines. It was also assumed that all male and female selection candidates from the 1000 sow maternal line nucleus herds were genotyped at low density and all animals used for breeding at high density. With the assumptions used in this analysis, it was estimated that genome-enabled selection costs for a maternal line would be approximately US$0.082 per weaned pig in the commercial production system. A total of US$0.164 per weaned pig is needed to incorporate genome-enabled selection into the two maternal lines. Similarly, for a 600 sow terminal line nucleus herd and genotyping only male selection candidates with the low-density panel, the cost per weaned pig in the commercial herd was estimated to be US$0.044. This means that US$0.21 per weaned pig produced at the commercial level and sired by boars obtained from the nucleus herd breeding program needs to be added to the genetic merit value in order to break even on the additional cost required when genome-enabled selection is used in both maternal lines and the terminal line.

Conclusions

By modifying the input values, such as herd size and genotyping strategy, a flexible spreadsheet tool developed from this work can be used to estimate the additional costs associated with genome-enabled selection. This tool will aid breeders in estimating the economic viability of incorporating genome-enabled selection into their specific breeding program.     

A computational approach to animal breeding

We propose a computational model of mating strategies for controlled animal breeding programs. A mating strategy in a controlled breeding program is a heuristic with some optimization criteria as a goal. Thus, it is appropriate to use the computational tools available for analysis of optimization heuristics. In this paper, we propose the first discrete model of the controlled animal breeding problem and analyse heuristics for two possible objectives: (1) breeding for maximum diversity and (2) breeding a target individual. These two goals are representative of conservation biology and agricultural livestock management, respectively. We evaluate several mating strategies and provide upper and lower bounds for the expected number of matings. While the population parameters may vary and can change the actual number of matings for a particular strategy, the order of magnitude of the number of expected matings and the relative competitiveness of the mating heuristics remains the same. Thus, our simple discrete model of the animal breeding problem provides a novel viable and robust approach to designing and comparing breeding strategies in captive populations.     

A method for non-invasive genotyping of APCmin/+ mice using fecal samples

The APC^min/+ mouse is commonly used in cancer research and is just one of many genetically altered models that is currently being developed. With high numbers of breeding programs, it is important to have a simple method that can be used to genotype the mice non-invasively. Here we report a reproducible method for genotyping mice with DNA extracted from fecal samples. Comparison of fecal results with those obtained from intestinal tissue DNA and clinical outcome (presence/absence of tumors) showed this technique to have 100% accuracy. This non-invasive method of genotyping may be applied to other transgenic mouse models.     

Genotype, phenotype, and karyotype correlation in the XO mouse model of Turner Syndrome

The murine model for Turner Syndrome is the XO mouse. Unlike their human counterparts, XO mice are typically fertile, and their lack of a second sex chromosome can be transmitted from one generation to the next as an X-linked dominant trait with male lethality. The introduction of an X-linked coat-color marker (tabby) has greatly facilitated the maintenance of this useful mouse strain. XO mice can be produced in large numbers, generation after generation, and rapidly identified on the basis of their sex and coat color. Although this breeding scheme appears to be effective at the phenotype level, its utility has never been conclusively proved at the molecular or cytogenetic levels. Here, we clone and sequence the tabby deletion break point and present a multiplex polymerase chain reaction-based assay for the tabby mutation. By combining the results of this assay with whole-chromosome painting data, we demonstrate that genotype, phenotype, and karyotype all show perfect correlation in the publicly available XO breeding stock. This work lays the foundation for the use of this strain to study Turner Syndrome in particular and the X chromosome in general.     

Generation of Rm21LG transgenic mice: a powerful tool to generate conditional overexpression of miR-21 that is involved in oncogenesis

miR-21 is highly expressed in a variety of cancers, suggesting that it might play a role in the process of oncogenesis, as supported by it directly causing pre-B cell lymphomas in transgenic mice. Rm21LG transgenic mice for the conditional co-expression of miR-21 and luciferase (Luc) mediated by Cre/lox P system were generated. The homozygous Rm21LG transgenic mice were visually and readily characterized immediately after birth by whole-body fluorescence imaging. More importantly, miR-21 and Luc were successfully activated in the liver of Rm21LG/Alb-Cre double-transgenic mice, demonstrating that Rm21LG conditional transgenic system could work in a Cre-dependent manner. The combined use of this conditional miR-21 transgenic mouse line, various cell/tissue-specific Cre mouse lines and bioluminescence imaging will be a valuable tool in vivo to uncover the functions of miR-21 as oncomiR in initiating tumors.     

Generation of mice with a conditional allele of the p120 Ras GTPase-activating protein

p120 Ras GTPase-activating protein (RasGAP) encoded by the rasa1 gene in mice is a prototypical member of the RasGAP family of proteins involved in negative-regulation of the p21 Ras proto-oncogene. RasGAP has been implicated in signal transduction through a number of cell surface receptors. In humans, inactivating mutations in the coding region of the RASA1 gene cause capillary malformation arteriovenous malformation. In mice, generalized disruption of the rasa1 gene results in early embryonic lethality associated with defective vasculogenesis and increased apoptosis of neuronal cells. The early lethality in this mouse model precludes its use to further study the importance of RasGAP as a regulator of cell function. Therefore, to circumvent this problem, we have generated a conditional rasa1 knockout mouse. In this mouse, an exon that encodes a part of the RasGAP protein essential for catalytic activity has been flanked by loxP recognition sites. With the use of different constitutive and inducible Cre transgenic mouse lines, we show that deletion of this exon from the rasa1 locus results in effective loss of expression of catalytically-active RasGAP from a variety of adult tissues. The conditional rasa1 mouse will be useful for the analysis of the role of RasGAP in mature cell types.     

General or cell type-specific deletion and replacement of connexin-coding DNA in the mouse

Here we describe several gene targeting approaches currently used in our laboratory for the generation of deletion or replacement mutants of connexin genes in the mouse and discuss the advantage of the double-replacement strategy for the generation of conditional mutants. For the analysis of complementary functions of connexins, it will be necessary to generate mice with mutations in several connexin genes. We also report how this can be effectively accomplished. The replacement of targeted connexin-coding DNA with a reporter gene, to mimic expression of the deleted gene product, is currently being used in several laboratories. The use of different reporter genes or their differently localized gene products could allow distinction of promoter activity in double or triple connexin mutant mice.     

High throughput gene trapping and postinsertional modifications of gene trap alleles

Gene trapping is a high-throughput insertional mutagenesis approach that has been primarily used in mouse embryonic stem cells (ESCs). As a high throughput technology, gene trapping helped to generate tenth of thousands of ESC lines harboring mutations in single genes that can be used for making knock-out mice. Ongoing international efforts operating under the umbrella of the International Knockout Mouse Consortium (IKMC; www.knockoutmouse.org) aim to generate conditional alleles for every protein coding gene in the mouse genome by high throughput conditional gene targeting and trapping. Here, we provide protocols for gene trapping in ESCs that can be easily adapted to any other mammalian cell. We further provide protocols for handling and verifying conditional gene trap alleles in ESC lines obtained from the IKMC repositories and describe a highly efficient method for the postinsertional modification of gene trap alleles. More specifically, we describe a protein tagging strategy based on recombinase mediated cassette exchange (RMCE) that enables protein localization and protein-protein interaction studies under physiological conditions.     

Rapid generation of inducible mouse mutants

We have generated an optimized inducible recombination system for conditional gene targeting based on a Cre recombinase–steroid receptor fusion. This configuration allows efficient Cre-mediated recombination in most organs of the mouse upon induction, without detectable background activity. An ES cell line, was established that carries the inducible recombinase and a loxP-flanked lacZ reporter gene. Out of this line, completely ES cell-derived mice were efficiently produced through tetraploid blastocyst complementation, without the requirement of mouse breeding. Our findings provide a new concept allowing the generation of inducible mouse mutants within 6 months, as compared to 14 months using the current protocol.     

A class of Bayesian methods to combine large numbers of genotyped and non-genotyped animals for whole-genome analyses

Background

To obtain predictions that are not biased by selection, the conditional mean of the breeding values must be computed given the data that were used for selection. When single nucleotide polymorphism (SNP) effects have a normal distribution, it can be argued that single-step best linear unbiased prediction (SS-BLUP) yields a conditional mean of the breeding values. Obtaining SS-BLUP, however, requires computing the inverse of the dense matrix G of genomic relationships, which will become infeasible as the number of genotyped animals increases. Also, computing G requires the frequencies of SNP alleles in the founders, which are not available in most situations. Furthermore, SS-BLUP is expected to perform poorly relative to variable selection models such as BayesB and BayesC as marker densities increase.

Methods

A strategy is presented for Bayesian regression models (SSBR) that combines all available data from genotyped and non-genotyped animals, as in SS-BLUP, but accommodates a wider class of models. Our strategy uses imputed marker covariates for animals that are not genotyped, together with an appropriate residual genetic effect to accommodate deviations between true and imputed genotypes. Under normality, one formulation of SSBR yields results identical to SS-BLUP, but does not require computing G or its inverse and provides richer inferences. At present, Bayesian regression analyses are used with a few thousand genotyped individuals. However, when SSBR is applied to all animals in a breeding program, there will be a 100 to 200-fold increase in the number of animals and an associated 100 to 200-fold increase in computing time. Parallel computing strategies can be used to reduce computing time. In one such strategy, a 58-fold speedup was achieved using 120 cores.

Discussion

In SSBR and SS-BLUP, phenotype, genotype and pedigree information are combined in a single-step. Unlike SS-BLUP, SSBR is not limited to normally distributed marker effects; it can be used when marker effects have a t distribution, as in BayesA, or mixture distributions, as in BayesB or BayesC π. Furthermore, it has the advantage that matrix inversion is not required. We have investigated parallel computing to speedup SSBR analyses so they can be used for routine applications.

Electronic supplementary material

The online version of this article (doi:10.1186/1297-9686-46-50) contains supplementary material, which is available to authorized users.     

Hybrid embryonic stem cell-derived tetraploid mice show apparently normal morphological,physiological, and neurological characteristics

ES cell-tetraploid (ES) mice are completely derived from embryonic stem cells and can be obtained at high efficiency upon injection of hybrid ES cells into tetraploid blastocysts. This method allows the immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps. To provide a baseline for the analysis of ES mouse mutants, we performed a phenotypic characterization of wild-type B6129S6F(1) ES mice in relation to controls of the same age, sex, and genotype raised from normal matings. The comparison of 90 morphological, physiological, and behavioral parameters revealed elevated body weight and hematocrit as the only major difference of ES mice, which exhibited an otherwise normal phenotype. We further demonstrate that ES mouse mutants can be produced from mutant hybrid ES cells and analyzed within a period of only 4 months. Thus, ES mouse technology is a valid research tool for rapidly elucidating gene function in vivo.     

Discovery genetics: serendipity in basic research

The role of serendipity in science has no better example than the discovery of spontaneous mutations that leads to new mouse models for research. The approach of finding phenotypes and then carrying out genetic analysis is called forward genetics. Serendipity is a key component of discovering and developing mice with spontaneous mutations into animal models of human disease. In this article, the role of serendipity in discovering and developing mouse models is described within a program at The Jackson Laboratory that capitalizes on serendipitous discoveries in large breeding colonies. Also described is how any scientists working with mice can take advantage of serendipitous discoveries as a research strategy to develop new models. Spontaneous mutations cannot be planned but happen in all research mouse colonies and are discovered as unexpected phenotypes. The alert scientist or technician can rationally exploit such chance observations to create new research opportunities.     

Genotype Selection to Rapidly Breed Congenic Strains

Congenic strains can now be constructed guided by the transmission of DNA markers. This allows not only selection for transmission of a desired, donor-derived differential region but also selection against the transmission of unwanted donor origin genomic material. The additional selection capacity should allow congenic strains to be produced in fewer generations than is possible with random backcrosses. Here, we consider modifications of a standard backcross breeding scheme to produce congenic mice by the inclusion of genotype-based selective breeding strategies. Simulation is used to evaluate the consequences of each strategy on the number of chromosomes that contain unwanted, donor-derived genetic material and the average length of this unwanted donor DNA for each backcross generation. Our prototypic strategy was to choose a single mouse to sire each generation using criteria designed to select against the transmission of chromosomes, other than the one containing the replacement genomic region, that contain any donor origin sequence at all. This chromosome elimination strategy resulted in an average of 16.4 chromosomes free of donor DNA in mice of the third backcross (N(3)) generation. A strategy based solely on positive selection for the replacement region required six backcross generations to achieve the same results.     

Optimum allocation of test resources and comparison of breeding strategies for hybrid wheat

Key message

The use of a breeding strategy combining the evaluation of line per se with testcross performance maximizes annual selection gain for hybrid wheat breeding.

Abstract

Recent experimental studies confirmed a high commercial potential for hybrid wheat requiring the design of optimum breeding strategies. Our objectives were to (1) determine the optimum allocation of the type and number of testers, the number of test locations and the number of doubled haploid lines for different breeding strategies, (2) identify the best breeding strategy and (3) elaborate key parameters for an efficient hybrid wheat breeding program. We performed model calculations using the selection gain for grain yield as target variable to optimize the number of lines, testers and test locations in four different breeding strategies. A breeding strategy (BS2) combining the evaluation of line per se performance and general combining ability (GCA) had a far larger annual selection gain across all considered scenarios than a breeding strategy (BS1) focusing only on GCA. In the combined strategy, the production of testcross seed conducted in parallel with the first yield trial for line per se performance (BS2_rapid) resulted in a further increase of the annual selection gain. For the current situation in hybrid wheat, this relative superiority of the strategy BS2_rapid amounted to 67 % in annual selection gain compared to BS1. Varying a large number of parameters, we identified the high costs for hybrid seed production and the low variance of GCA in hybrid wheat breeding as key parameters limiting selection gain in BS2_rapid.     

Invited review: genetic dissection of sleep.

Recent advances in genomics open up new avenues in the analysis of complex behaviors such as sleep. In this analysis, the mouse is the model species of choice because it is amenable to high throughput phenotype and genotype analysis. With the use of the mouse model, unprecedented progress in our understanding of sleep physiology and the treatment of sleep disorders is awaited. This review is intended to provide an overview of available methods and techniques for genetic dissection of sleep in mice. Limits and advantages of different approaches are discussed to highlight the necessity for combining methods to avoid erroneous interpretations. The gap between understanding mechanisms of sleep and its functions may be bridged by finding its molecular bases.