G1 Offspring Research Articles

Whole-Exome Sequencing to Identify Mutations in Thrombosis Modifier Genes Isolated From a Factor V Leiden-Dependent Sensitized ENU Suppressor Screen in the Mouse

Abstract 1183Only ∼10% of individuals carrying the common venous thrombosis risk factor, Factor V Leiden (FVL) will develop venous thrombosis in their lifetime. In order to identify potential FVL modifier genes, we performed a sensitized dominant ENU mutagenesis screen, based on the perinatal synthetic lethal thrombus previously observed in mice homozygous for FVL (FVQ/Q) and hemizygous for tissue factor pathway inhibitor deficiency (Tfpi+/−). The genome-wide ENU mutagenesis screen was performed by crossing ENU-treated male FVQ/Q mice with FVQ/+ Tfpi+/− females. Surviving G1 offspring were analyzed to identify survivors with the otherwise lethal FVQ/Q Tfpi+/− genotype. As proof of concept, we demonstrated that reduced tissue factor (Tf+/−) suppresses the lethal FVQ/Q Tfpi+/− phenotype, suggesting that mutations at Tf should be among the suppressor genes identified by our screen. Analysis of 7,128 G1 offspring (∼2X genome coverage) identified 98 FVQ/Q Tfpi+/− mice that survived to weaning. Fourteen FVQ/Q Tfpi+/− G1 mice exhibited successful transmission of a putative suppressor mutation to two or more FVQ/Q Tfpi+/− G2 offspring. Extensive genotyping of mice from an expanded genetic cross from one of these lines mapped a candidate suppressor locus to a chromosome 3 region encompassing the TF gene (LOD=4.93). With continued improvements in next generation sequencing technologies, we have now applied whole exome sequencing to analysis of 8 of the remaining 13 lines. The entire DNA coding region (the “exome”, totaling 49.6 Mb of DNA sequence) from a progeny-tested member of each line was captured using the Agilent SureSelect mouse exome capture system. Whole-exome sequencing using the Illumina HiSeq high-throughput sequencer yielded 12–15 gigabases of sequence data per sample, corresponding to an average of ∼200 fold sequencing coverage for each nucleotide position. Variant analysis using the Gene Analysis Toolkit revealed the presence of a small number of high confidence novel heterozygous (dominant) variants in each sample. Each of these heterozygous variants is a candidate suppressor mutation and these are presently being tested in remaining FVQ/Q Tfpi+/− mice from each respective line. Based on previous studies where ENU-induced mutations in mice have been identified, we anticipate the identification of putative exomic mutations in approximately 80% of tested suppressor lines. Identification of these mutations should provide candidate modifier genes for FVL and other human hemostatic disorders. Disclosures:Ginsburg:Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Catalyst Biosciences: Membership on an entity's Board of Directors or advisory committees; Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Effects of Passiflora edulis on the Metabolic Profile of Diabetic Wistar Rat Offspring

Dry extract of the genus Passiflora has been shown to help control glycemia and lipid levels. The objective of this study was to evaluate the effects of passion fruit (P. edulis) on the biochemical profile of offspring from diabetic rats. Diabetes was induced by streptozotocin. The diabetes group consisted of 10 rats with glucose levels greater than 200 mg/dL; the nondiabetic (control) group consisted of 10 rats with glucose levels less than 120 mg/dL. After the diagnosis of diabetes, the mating phase was started. By day 21 of pregnancy, the offspring were born; the dams were kept in individual cages with their offspring until the weaning period. The offspring were then divided into 4 groups (n=15 each): G1 were offspring from control dams, G2 were offspring from treated nondiabetic dams, G3 were offspring from diabetic dams, and G4 were offspring from treated diabetic dams. For 30 consecutive days, G1 and G3 offspring were treated with vehicle (oral gavage) and G2 and G4 offspring were treated with passion fruit juice (oral gavage). After 30-day treatment, the animals were anesthetized and killed, and blood was drawn immediately for analysis of the biochemical profile (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and glucose). The G2 and G4 rats showed significantly reduced total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels and an increased high-density lipoprotein cholesterol level. The use of passion fruit juice improved lipid profiles, suggesting that this plant may have beneficial effects in the prevention and treatment of dyslipidemias and hyperglycemia.

Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in hematological parameters

Research on hematological disorders relies on suitable animal models. We retrospectively evaluated the use of the hematological parameters hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell count (RBC), white blood cell count (WBC), and platelet count (PLT) in the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project as parameters for the generation of novel animal models for human diseases. The analysis was carried out on more than 16,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the levels of the chosen parameters. Identification of animals exhibiting altered values and transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters MCV, RBC, and PLT. Analysis of the causative mutation was started in selected lines, thereby revealing a novel mutation in the transferrin receptor gene (Tfrc) in one line. Thus, novel phenotype-driven mouse models were established to analyze the genetic components of hematological disorders.

The Role of Maternal Early-Life and Later-Life Risk Factors on Offspring Low Birth Weight: Findings From a Three-Generational Study

This study examined three research questions: (1) Is there an association between maternal early-life economic disadvantage and the birth weight of later-born offspring? (2) Is there an association between maternal abuse in childhood and the birth weight of later-born offspring? (3) To what extent are these early-life risks mediated through adolescent and adult substance use, mental and physical health status, and adult socioeconomic status (SES)? Analyses used structural equation modeling to examine data from two longitudinal studies, which included three generations. The first generation (G1) and the second generation (G2) were enrolled in the Seattle Social Development Project (SSDP), and the third generation (G3) was enrolled in the SSDP Intergenerational Project. Data for the study (N = 136) focused on (G2) mothers enrolled in the SSDP and their children (G3). Analyses revealed that G2 low childhood SES predicted G3 offspring birth weight. Early childhood abuse among G2 respondents predicted G3 offspring birth weight through a mediated pathway including G2 adolescent substance use and G2 prenatal substance use. Birth weight was unrelated to maternal adult SES, depression, or obesity. To our knowledge, this is the first study to identify the effect of maternal early-life risks of low childhood SES and child maltreatment on later-born offspring birth weight. These findings have far-reaching effects on the cumulative risk associated with early-life economic disadvantage and childhood maltreatment. Such findings encourage policies and interventions that enhance child health at birth by taking the mother's own early-life and development into account.

Generation of N‐ethyl‐N‐nitrosourea‐induced mouse mutants with deviations in plasma enzyme activities as novel organ‐specific disease models

Measurement of plasma enzyme activities is part of routine medical examination protocols and provides valuable parameters for the diagnosis of various organ diseases. In the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project, clinical chemical blood analysis was carried out on more than 20,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma enzyme activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, alpha-amylase and creatine kinase. We identified a large number of animals that consistently exhibited altered plasma enzyme activities. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for each parameter. Breeding experiments in selected lines detected the linkage of the causative mutations to defined chromosomal regions. Subsequently, identification of the mutated genes was successfully carried out in chosen lines, resulting in a novel alkaline phosphatase liver/bone/kidney (Alpl) alteration in one line and the strong indication for a dystrophin (Dmd) alteration in another line. The mouse mutants with abnormal plasma enzyme activities recovered in the Munich ENU project are novel tools for the systematic dissection of the pathogenesis of organ diseases.

N-ethyl-N-nitrosourea mutagenesis produced a small number of mice with altered plasma electrolyte levels

BackgroundClinical chemical blood analysis including plasma electrolytes is routinely carried out for the diagnosis of various organ diseases. Phenotype-driven N-ethyl-N-nitrosourea (ENU) mouse mutagenesis projects used plasma electrolytes as parameters for the generation of novel animal models for human diseases.MethodsHere, we retrospectively evaluated the use of the plasma electrolytes calcium, chloride, inorganic phosphorus, potassium and sodium in the Munich ENU mouse mutagenesis project where clinical chemical blood analysis was carried out on more than 20,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in various plasma parameter levels.ResultsWe identified a small number of animals consistently exhibiting altered plasma electrolyte values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters calcium and potassium. Published data from other phenotype-driven ENU projects also included only a small number of mutant lines which were generated according to altered plasma electrolyte levels.ConclusionThus, use of plasma electrolytes detected few mouse mutants in ENU projects compared to other clinical chemical blood parameters.

Sobrepeso na adolescência de filhos de mães que tiveram distúrbios glicêmicos na gestação

To verify whether different hyperglycemia levels during pregnancy cause frequency differences in adolescent obesity and its morbidities in the offspring. Seventy-three children were divided into three groups according to maternal glucose tolerance: G1 (n=27) normal oral glucose tolerance test (OGTT) and daily glycemia (DG); G2 (n=23) normal OGTT and high DG; G3 (n=23) abnormal OGTT and DG (gestational diabetes mellitus; GDM). All underwent clinical evaluation (anthropometry) a questionnaire(neonatal data, eating habits), and determination of fasting serum glucose and lipid profile measurement. Analysis of variance (ANOVA) and the Goodmans test were used to compare the groups. G3 mothers showed higher fasting plasma glucose(FPG) and DG than G2 and G1(FPG: 93+/-10 vs 83+/-5 vs 78+/-10 mg/dL; DG: 104+/-12 vs 93+/-7 vs 85+/-9 mg/dL, respectively; P<0.001). G2 mothers had higher DG than G1 (93+/-7 vs 85+/-9 mg/dL; P<0.001). G3 offspring birthweight was higher than G1 and G2 (3,667+/-527 vs 3,167+/-565 and 3,282+/-401 g, respectively; P<0.05). More G3 offspring were overweight than G1 (52.2 vs 14.8%; P<0.05). Offspring of GDM mothers with fasting and daily hyperglycemia have higher birthweight and overweight frequency in adolescence. These children must be followed up from infancy.

Gene-environment interactions in a mutant mouse kindred with native airway constrictor hyperresponsiveness

We mutagenized male BTBR mice with N-ethyl-N-nitrosourea and screened 1315 of their G3 offspring for airway hyperresponsiveness. A phenovariant G3 mouse with exaggerated methacholine bronchoconstrictor response was identified and his progeny bred in a nonspecific-pathogen-free (SPF) facility where sentinels tested positive for minute virus of mice and mouse parvovirus and where softwood bedding was used. The mutant phenotype was inherited through G11 as a single autosomal semidominant mutation with marked gender restriction, with males exhibiting almost full penetrance and very few females phenotypically abnormal. Between G11 and G12, facility infection eradication was undertaken and bedding was changed to hardwood. We could no longer detect airway hyperresponsiveness in more than 37 G12 offspring of 26 hyperresponsive G11 males. Also, we could not identify the mutant phenotype among offspring of hyperresponsive G8-G10 sires rederived into an SPF facility despite 21 attempts. These two observations suggest that both genetic and environmental factors were needed for phenotype expression. We suspect that rederivation into an SPF facility or altered exposure to pathogens or other unidentified substances modified environmental interactions with the mutant allele, and so resulted in disappearance of the hyperresponsive phenotype. Our experience suggests that future searches for genes that confer susceptibility for airway hyperresponsiveness might not be able to identify some genes that confer susceptibility if the searches are performed in SPF facilities. Experimenters are advised to arrange for multigeneration constancy of mouse care in order to clone mutant genes. Indeed, we were not able to map the mutation before losing the phenotype.

Characterization of a donor mitochondrial DNA transmission bottleneck in nuclear transfer derived cow lineages

In embryos derived by nuclear-transfer (NT), fusion of donor cells with recipient oocytes resulted in varying patterns of mitochondrial DNA (mtDNA) transmission in NT animals. Distribution of donor cell mtDNA (D-mtDNA) found in offspring of NT-derived founders may also vary from donor cell and host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to G(1) offspring. Eleven NT founder cows were produced by fusion of enucleated oocytes (Holstein/Japanese Black) with Jersey/ Holstein oviduct epithelial cells, or Holstein/Japanese Black cumulus cells. Transmission of mtDNA was analyzed by PCR mediated single-strand conformation polymorphism of the D-loop region. In six of seven animals sampled postmortem, heteroplasmy were detected in various tissues, while D-mtDNA could not be detected in blood or hair samples from four live animals. The average proportion of D-mtDNA detected in one NT cow was 7.6%, and those in other cows were <5%. Heteroplasmic NT cows (n = 6) generated a total 12 G(1) offspring. Four of 12 G(1) offspring exhibited high percentages of D-mtDNA populations (range 17-51%). The remaining eight G(1) offspring had slightly or undetectable D-mtDNA (<5%). Generally, a genetic bottleneck in the female germ-line should favor a homoplasmic state. However, proportions of some G(1) offspring maintained heteroplasmy with a much higher percentage of D-mtDNA than their NT dams, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrate that D-mtDNA in NT cows is transmitted to G(1) offspring with varying efficiencies.

INDUCTION OF TETRAPLOIDY IN TRANSGENIC TILAPIA (OREOCHROMIS NILOTICUS) USING PHYSICAL SHOCKS

The induction of tetraploidy by means of cold, heat and multiple heat shock treatments was investigated on male fish from a growth-enhanced transgenic tilapia C118 line, crossed with wild type females. After the development a new multiple heat shock protocol (two heat treatments at 41oC in 60 and 80 min. after fertilization for 5 min. per each), chromosome and X-gal in situ staining assays demonstrated the successful production of yolk sac tetraploid transgenic tilapia in two out of 15 embryos examined in this trial via direct correlation between higher level of b-galactosidase expression and karyotyping in the samples verifying complete tetraploidy in this line. In the grown fish, there was evidence to suggest mosaicism or perhaps tetraploidy in at least two grown fish using the measurement of erythrocytes and karyotyping of the G1 offspring (P<0.05). There was, however, no evidence to indicate successful production of triploid G1 individuals following crossing of induced fish with wild type.

AN ENU MUTAGENESIS SCREEN FOR DOMINANT GENETIC MODIFIERS OF THROMBOSIS IN THE FACTOR 5 LEIDEN MOUSE

ISTH07A1_P-W-473: Contact View [P-W-473] AN ENU MUTAGENESIS SCREEN FOR DOMINANT GENETIC MODIFIERS OF THROMBOSIS IN THE FACTOR 5 LEIDEN MOUSE R.J. Westrick, S.L. Manning, M.E. Winn, G.M. Stotz, E. Sanford, D. Ginsburg. Human Genetics; Howard Hughes Medical Institute; Internal Medicine; Life Sciences Institute, University of Michigan, Ann Arbor, United States Introduction: Factor V Leiden, (F5Q) is the most common inherited risk factor for venous thrombosis. However, a relatively small percentage of people carrying F5Q will develop venous thrombosis in their lifetimes. This variability results from complex interactions between the environment and unknown genetic factors (modifier genes). We previously identified a synthetically lethal interaction between mice inheriting two copies of F5Q (F5Q/Q) and partial Tfpi (Tfpi+/-) deficiency (F5Q/Q Tfpi+/-). Complete Tfpi deficiency in mice is embryonic lethal, whereas heterozygosity is compatible with normal survival. However, all mice inheriting F5Q/Q and Tfpi+/die of thrombosis shortly after birth. Methods: The F5Q/Q Tfpi+/lethal interaction was utilized as the basis for a sensitized ENU mutagenesis screen designed to uncover novel dominant suppressor mutations that alter the hemostatic balance. Male F5Q/Q mice were ENU mutagenized and bred to F5Q/+ Tfpi+/double heterozygous females. Surviving offspring were analyzed to identify rescued mice with the F5Q/Q Tfpi+/genotype. Results: Analysis of 5600 G1 offspring to date has identified 64 mice that survived to weaning. Of the 12 F5Q/Q Tfpi+/mutants tested for heritability, five produced additional F5Q/Q Tfpi+/progeny, demonstrating that the associated thrombosis suppressor is heritable. The latter progeny have been genotyped with a panel of markers spanning the genome. For one of these mutant lines, this analysis has mapped the corresponding suppressor mutation to a small region on Chromosome 9, with fine mapping to identify the corresponding gene underway. Conclusions: We have demonstrated the existence of novel dominant mutations that improve hemostatic balance leading to survival of F5Q/Q Tfpi+/mice. Characterization of the genes may provide novel insight into the regulation of hemostasis, as well as identifying potential candidates for thrombosis modifiers in humans. Westrick RJ, Manning SL, Winn ME, Stotz GM, Sanford E, Ginsburg D. AN ENU MUTAGENESIS SCREEN FOR DOMINANT GENETIC MODIFIERS OF THROMBOSIS IN THE FACTOR 5 LEIDEN MOUSE. J Thromb Haemost 2007; 5 Supplement 2: P-W-473 Date: Wednesday, July 11, 2007 Session Info: Poster: Thrombophilia and familial thrombosis Room: Exhibition Area file:///E|/working/RAJAN/Wednesday/P-W-473.htm [1/22/2014 3:39:03 PM]

Generation of ENU‐Induced Mouse Mutants with Hypocholesterolemia: Novel Tools for Dissecting Plasma Lipoprotein Homeostasis

Pathologic plasma lipoprotein cholesterol levels play a key role in the development and pathogenesis of human atherosclerotic cardiovascular diseases. Plasma cholesterol homeostasis is regulated by genetic predispositions and environmental factors. Animal models showing aberrant plasma cholesterol levels are used for the identification and analysis of novel causative genes. Here, we searched for inherited hypocholesterolemia phenotypes in randomly mutant mice which may contribute to the detection of disease protective alleles. In the Munich ENU mouse mutagenesis project, clinical chemistry blood analysis was carried out on more than 15,500 G1 offspring and 230 G3 pedigrees of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to a decreased plasma total cholesterol level. We identified 66 animals consistently showing hypocholesterolemia. Transmission of the altered phenotype to the subsequent generations led to the successful establishment of 14 independent hypocholesterolemic lines. Line-specific differences were detected by clinical chemistry analysis of plasma HDL cholesterol, LDL cholesterol and triglycerides. Thus, we successfully established a novel panel of ENU-derived mutant mouse lines for their use in the identification of alleles selectively influencing the plasma cholesterol homeostasis. Such findings may be subsequently used for humans and other species.

Screening for increased plasma urea levels in a large-scale ENU mouse mutagenesis project reveals kidney disease models

Kidney diseases lead to the failure of urinary excretion of metabolism products. In the Munich ethylnitrosourea (ENU) mouse mutagenesis project, which is done on a C3H inbred genetic background, blood samples of more than 15,000 G1 offspring and 500 G3 pedigrees were screened for alterations in clinical-chemical parameters. We identified 44 animals consistently exhibiting increased plasma urea concentrations. Transmission analysis of the altered phenotype of 23 mice to subsequent generations led to the establishment of five mutant lines. Both sexes were affected in these lines. Urinary urea levels were decreased in the mutants. In addition, most mutants showed increased plasma and decreased urinary creatinine levels. Pathological investigation of kidneys from the five mutant lines revealed a broad spectrum of alterations, ranging from no macroscopic and light microscopic kidney alterations to decreased kidney weight-to-body weight ratio, dilation of the renal pelvis, and severe glomerular lesions. Thus screening for elevated plasma urea levels in a large-scale ENU mouse mutagenesis project resulted in the successful establishment of mouse strains which are valuable tools for molecular studies of mechanisms involved in urea excretion or which represent interesting models for kidney diseases.

379 LACTATION INDUCTION IN PREPUBERTAL BULLS AND HEIFERS AS A TOOL FOR PREDICTING MAMMARY SPECIFIC TRANSGENE EXPRESSION IN CATTLE

The bovine's long generation interval results in a lapse, from the time of birth, of two to three years before mammary-specific transgenes can be assessed in genetically engineered animals. This experiment was conducted in an attempt to reduce that waiting period by up to two years. Lactation was induced in prepubertal bull and heifer calves, 3 to 8 mo of age, as a means of predicting transgene behavior during subsequent normal lactations in the heifers and daughters of bulls. Transgenic animals tested were either founder animals, produced by somatic cell nuclear transfer, or G1 offspring of founder bulls. The transgene consists of a lactation specific sequence encoding lysostaphin, an antimicrobial protein targeted against Staphylococcus aureus, a mastitis-causing pathogen. Estrogen, progesterone, and dexamethasone were administered as previously described (Ball et al. 2000 J. Dairy Sci. 83, 2459) to nine heifers (transgenics = 5) ranging in weight from 90 to 165 kg. Eight bull calves (transgenics = 7) weighing from 81 to 178 kg received additional estrogen and progesterone injections as well as reserpine prior to dexamethasone treatment. Animals were hand-milked twice daily for 4 to 7 days. All nine heifers responded to the milk induction scheme, yielding between 19 mL and 4.5 L. Milk volume from the three responding males (100 �L to 2.5 mL) was significantly less than that harvested from females (P = 0.025). Only bull calves over 150 kg had a positive response. Transgenic females produce less milk then non-transgenics (313 � 494 vs. 2276 � 552 mL, respectively; P = 0.033). Most importantly, there was no detectable difference between the concentration of lysostaphin in milk from induction (8.1 � 2.7 �g/mL) and natural lactations (3.5 � 2.6 �g/mL) in the four transgenic heifers tested (P = 0.229). The result was the same when lysostaphin was analyzed as a percentage of total protein (P = 0.427). Induction of a G1 heifer and a bull calf from the same founder bull produced similar lysostaphin concentrations in their milk (5.6 � 0.9 and 5.2 � 0.5 �g/mL; respectively). �-lactoglobulin concentration was also similar during induced and natural lactation (P = 0.165) for all animals studied. However, total protein was greater in induced milk samples compared to natural lactation samples (28.4 � 1.7 vs. 21.2 � 1.7 mg/mL; P = 0.007) as was lactoferrin (707 � 51 vs. 213 � 51 �g/mL; P &lt; 0.001). Conversely, compared to that in induced milk samples, lactose was more concentrated in the natural lactation samples (34.6 � 2.5 vs. 46.0 � 2.1 g/L). In this study transgene expression was detected in milk from induced lactations and its concentration in those samples was generally predictive of product concentration in the natural lactation milk. The induction protocol was effective in male (&gt;150 kg) and female calves.

Production of Transgenic Quails with High Frequency of Germ-Line Transmission Using VSV-G Pseudotyped Retroviral Vector

We report here the production of transgenic quails using a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus (MoMLV) pseudotyped with vesicular stomatitis virus G protein (VSV-G). The retroviral vector was injected into laid quail embryos at the blastodermal stage, and the embryos were incubated to hatch to produce G0 transgenic quails. Among 134 embryos subjected to viral injection, 37 hatched. The viral vector sequence was detected in the tissues of all G0 quails. The germ-line transmission efficiency of G0 quails mated with nontransgenic quails was more than 80% on average. Southern blot analysis revealed that the G1 transgenic progeny had one to three copies of the transgene. The expression of vector-encoded neomycin-resistance gene under the control of the Rous sarcoma virus (RSV) promoter was observed in several tissues including heart and muscle of both G1 and G2 transgenic offspring. Due to the high frequency of germ-line transmission, this method may markedly facilitate the production of transgenic avian.

A genetic screen for novel behavioral mutations in mice.

A genetic screen using mice was performed to identify dominant loci affecting behavior. Mice were mutagenized with ENU, then bred to examine their G1 offspring for behavioral abnormalities. Potentially mutant G1 pups were screened through a variety of behavioral assays, including tests of learning and memory, sensorimotor gating, fear and anxiety, nociception (pain perception) and locomotor activity. Mice falling outside the normal performance distribution in these tests were considered potential behavioral mutants and were bred for further analysis. Outliers included both animals with very discrete defects and animals with abnormal performance across a range of tests. To date, we have identified two confirmed mutants affecting sensorimotor gating. These results provide further impetus for the use of random mutagenesis screens as a tool for dissecting the genetic basis of brain and behavior.

Ballistic transfection of avian primordial germ cellin ovo

In the fowl the primordial germ cells accumulate in the germinal crescent to the anterior of the two-day embryo. A simple ballistic device has been used to fire tungsten particles (mean diameter 1.5 microns) into this region. By coating these projectiles with vector DNA it is possible to transfect these cells. Hatchlings produced by this technique were raised to sexual maturity and shown to contain the foreign DNA in their sperm. G1 offspring containing this DNA were also produced in roughly 20% of these cockerels. In the majority of cases the vector DNA disappeared from the G1 generation as they matured suggesting that in these cases it had been transmitted episomally.

Germline Transmission of Exogenous Genes in the Chicken

Difficulties associated with in vitro manipulation and culture of the early chicken embryo have restricted generation of transgenic chickens to approaches that use replication-competent retroviruses. The need to produce transgenic chickens in the absence of replicating virus prompted development of a new method of gene transfer into the chicken. Microinjection of the replication-defective reticuloendotheliosis virus (REV) vector ME111 beneath unincubated chicken embryo blastoderms results in infection of germline stem cells. This vector contains genetic information exogenous to the chicken genome, including both the herpes simplex virus type 1 thymidine kinase gene and the Tn5 neomycin phosphotransferase gene. About 8 percent of male birds hatched from injected embryos contained vector DNA in their semen. All four positive males tested passed vector sequences onto their progeny. Analysis of G1 offspring showed that gonads of G0 male birds were mosaic with respect to insertion of vector provirus. Thus, primordial germ cells present in the unincubated chicken embryo blastoderm are susceptible to infection by defective REV vectors.

Reproduction and Growth following Prenatal Methylmercuric Chloride Exposure in Mice

Reproduction and Growth following Prenatal Methylmercuric Chloride Exposure in Mice. GATES, A.H., DOHERTY, R.A., AND Cox, C. (1986). Fundam. Appl. Toxicol. 7, 486-493. Reproductive effects of the environmental pollutant methylmercuric chloride administered as a single dose per os during pregnancy were studied both in treated mice (Go generation) and in their prenatally exposed offspring (G1). Treatment at 9.5 days postfertilization caused no observed effects at doses as high as 12-18 mgHg+/kg. In contrast, treatment at 12.5 and 15.5 days produced toxicity; results were similar and were combined for analysis. Among Go mice, the percentage capable of delivering one or more viable pups showed an estimated threshold level response at 8.0 mg/kg. The percentage of G, pups that were viable at 1 day post partum was significantly dose-related, with an approximate threshold exposure level of 4.3 mg/kg. Among the surviving G1 offspring, body weight in adulthood (8 months) showed a statistically significant reduction that was also dose-related. Fertility testing of Gi offspring revealed no treatment effects on mating behavior judged by time interval between pairing and parturition. However, there was a trend (statistically nonsignificant) toward a dose effect on sizes of females' litters. Sterility (inability to produce offspring) did not occur either among Gi males at 4 months of age (N = 30; dose 8-12 mg/kg) or among females up to the advanced reproductive age of 14 months (N = 29; dose 5.3-12 mg/kg). We conclude that mice exposed prenatally to methylmercuric chloride revealed no greater susceptibility to sterility than to perinatal mortality.