Jeffrey R. Sommer

Transgenic animals as models for human disease.

Transgenic animals, especially mice, have been used quite extensively as models for various human diseases. At first, the level of scientific inquiry was driven by the need to establish the model. In many cases, these models may be considered quite crude because of their limitations. More recently, transgenic models of disease have become more refined and are currently being used to study the pathological mechanisms behind the disease rather than to just provide a model of the disease. Using some examples from the recent literature, we will document the current level and complexity of inquiry using transgenic animals. New techniques and techniques that may prove promising will be discussed.

Production of ELOVL4 transgenic pigs: a large animal model for Stargardt-like macular degeneration

Background Truncation mutations in the elongation of very long chain fatty acids-4 (AF277094, MIM #605512) (ELOVL4) gene cause Stargardt-like macular dystrophy type 3 (STGD3). Mice expressing truncated ELOVL4 develop rapid retinal degeneration, but are poor STGD3 models since mice lack a macula. Photoreceptor topography in the pig retina is more similar to that in humans as it includes the cone rich, macula-like area centralis. The authors generated transgenic pigs expressing human disease-causing ELOVL4 mutations to better model the pathobiology of this macular disease. Methods Pronuclear DNA microinjection and somatic cell nuclear transfer were used to produce transgenic pigs for two different ELOVL4 mutations: the 5 base pair deletion (5 bpdel) and the 270 stop mutation (Y270terEYFP). Retinal transgene expression, morphology and electrophysiology were examined. Results The authors obtained four lines of Y270terEYFP and one line of 5 bpdel transgenic animals. Direct fluorescence microscopy indicated that the Y270terEYFP protein is expressed in photoreceptors and mislocalised within the cell. Immunohistochemical examination of transgenic pigs showed photoreceptor loss and disorganised inner and outer segments. Electroretinography demonstrated diminished responses in both transgenic models. Conclusions These transgenic pigs provide unique animal models for examining macular degeneration and STGD3 pathogenesis.

Conditional Induction of Ovulation in Mice

Abstract Follicle-stimulating hormone controls the maturation of mammalian ovarian follicles. In excess, it can increase ovulation (egg production). Reported here is a transgenic doxycycline-activated switch, tested in mice, that produced more FSHB subunit (therefore more FSH) and increased ovulation by the simple feeding of doxycycline (Dox). The transgenic switch was expressed selectively in pituitary gonadotropes and was designed to enhance normal expression of FSH when exposed to Dox, but to be regulated by all the hormones that normally control FSH production in vivo. Feeding maximally effective levels of Dox increased overall mRNA for FSHB and serum FSH by over half in males, and Dox treatment more than doubled the normal ovulation rate of female mice for up to 10 reproductive cycles. Lower levels of Dox increased the number of developing embryos by 30%. Ovarian structure and function appeared normal. In summary, gene switch technology and normal FSH regulation were combined to effectively enhance ovulation in mice. Theoretically, the same strategy can be used with any genetic switch to increase ovulation (or any highly conserved physiology) in any mammal.

Reporter system for the detection of in vivo gene conversion: changing colors from blue to green using GFP variants.

We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr66-His66). Cells that were either transiently or stably transfected with the reporter system were used to test the functionality and feasibility of the detection of in vivo gene correction. A thio-protected single-stranded oligonucleotide designed to convert the genotype of the blue variant to that of the EGFP variant by the correction of a single base pair was delivered to the reported cells using a variety of methodologies and strategies. Conversion events were easily observed using fluorescent microscopy because of the enhanced emission intensity and different spectra of the EGFP variant.

Supplementation of maternal diets with docosahexaenoic acid and methylating vitamins impacts growth and development of fetuses from malnourished gilts

Abstract Background Like many species, pregnant swine mobilize and repartition body nutrient stores during extreme malnutrition to support fetal development. Objective The objective of this study was to model chronic human maternal malnutrition and measure effects of methylating-vitamins (MVs, containing choline, folate, B-6, B-12, and riboflavin) and docosahexaenoic acid (DHA) supplementation on fetal growth and development. Methods Pregnant gilts (n = 24) were either fully nourished (2.0 kg/d) with a corn-plus-isolated-soy-protein basal diet (control) supplemented with MVs and DHA or nourishment was restricted throughout gestation. Basal diet fed to malnourished gilts was reduced progressively from 50% to 70% restriction (1.0 to 0.6 kg/d) and was supplemented following a 2 (±MVs) x 2 (±DHA) factorial design. Full-term c-sections were performed to assess impacts on low and normal birth weight (LBW/NBW) fetuses (n = 238). Results Body weight gain of malnourished gilts was 10% of full-fed control dams (P < 0.05), but offspring birth weight, length, girth, and percentage of LBW fetuses were not different between treatments. The number of pigs per litter was reduced by 30% in malnourished control dams. Fetal brain weights were reduced by 7% compared to positive controls (P < 0.05). Micronutrient supplementation to malnourished dams increased fetal brain weights back to full-fed control levels. Dams with DHA produced offspring with higher DHA concentrations in brain and liver (P < 0.05). Plasma choline concentration was 4-fold higher in fetuses from unsupplemented malnourished dams (P < 0.0001). Global DNA methylation status of fetuses from restricted dams was higher than in control fetuses, including brain, liver, heart, muscle, and placenta tissues (P < 0.05). Addition of DHA increased methylation in LBW fetal brains (P < 0.05). Conclusions Despite the mobilization of maternal stores, malnourished litters displayed reduced brain development that was fully mitigated by micronutrient supplementation. Severe maternal malnutrition increased global DNA methylation in several fetal tissues that was unaltered by choline and B-vitamin supplementation.

Nr4A1 mRNA regulation of FSH-induced meiotic resumption in bovine cumulus-oocyte complexes

Objective: 1) further define the time course required for transcription initiation in bovine cumulus-oocyte-complexes (COC); 2) determine the pattern of expression for Nr4A1 and Egr1 mRNAs in bovine COC; and, 3) reduce Nr4A1 mRNA expression using small interfering RNAs (siRNA) to determine the effect on breakdown of the germinal vesicle (GVBD) Methods: A series of experiments were performed to define the time required for transcription initiation during FSH-induced maturation in bovine COCs, determine the pattern of expression for candidate mRNAs during GVBD, and use RNAi to determine their potential role in GVBD by examining whether candidate-specific siRNA can reduce mRNA expression in bovine COCs and affect the occurrence of GVBD. Results: Transcription required for GVBD in bovine COC occurred as early as 30 min after culture initiation. Expression of Nr4A1 mRNA increased (P <0.05) at 30 min after culture initiation, consistent with the time of transcription initiation required for GVBD. Expression of Egr1 mRNA did not differ during culture. Expression of Nr4A1 mRNA was decreased (P <0.05) in COC cultured with 50nM siNr4A1 or with 120 µM of the transcriptional inhibitor DRB compared to controls. The proportion of COC undergoing GVBD at 9 hr of culture in FSH and non-specific siRNA (siNS) treat ment groups did not differ. However, fewer (P <0.05) COC underwent GVBD at 9 hr of culture when in the presence of DRB or 50nM siNr4A1 compared to controls. Conclusion: these data support a role for Nr4A1 in regulating FSH-mediated and transcription-dependent GVBD in bovine COC cultured in vitro.

High mobility group A2 (HMGA2) deficiency in pigs leads to dwarfism, abnormal fetal resource allocation, and cryptorchidism

Significance We show that mutations in HMGA2 affect fetal resource allocation, testis descent, and the size of pigs and provides a target for gene modification that can be used to modulate size in other mammalian species. This can have implications in agriculture as well as in the development of new strains of companion animals. In addition, most xenograft pig donors have adult organs larger than those of humans. Recently, it has been shown that regulation of organ growth is donor-controlled, not host-controlled, resulting in organ overgrowth and damage after transplantation. We show here that the HMGA2 gene is a potential target for organ-size regulation in xenotransplantation. Expression of HMGA2 is strongly associated with body size and growth in mice and humans. In mice, inactivation of one or both alleles of Hmga2 results in body-size reductions of 20% and 60%, respectively. In humans, microdeletions involving the HMGA2 locus result in short stature, suggesting the function of the HMGA2 protein is conserved among mammals. To test this hypothesis, we generated HMGA2-deficient pigs via gene editing and somatic cell nuclear transfer (SCNT). Examination of growth parameters revealed that HMGA2−/+ male and female pigs were on average 20% lighter and smaller than HMGA2+/+ matched controls (P < 0.05). HMGA2−/− boars showed significant size reduction ranging from 35 to 85% of controls depending on age (P < 0.05), and organ weights were also affected (P < 0.05). HMGA2−/+ gilts and boars exhibited normal reproductive development and fertility, while HMGA2−/− boars were sterile due to undescended testes (cryptorchidism). Crossbreeding HMGA2−/+ boars and gilts produced litters lacking the HMGA2−/− genotype. However, analysis of day (D) D40 and D78 pregnancies indicated that HMGA2−/− fetuses were present at the expected Mendelian ratio, but placental abnormalities were seen in the D78 HMGA2−/− concepti. Additionally, HMGA2−/− embryos generated by gene editing and SCNT produced multiple pregnancies and viable offspring, indicating that lack of HMGA2 is not lethal per se. Overall, our results show that the effect of HMGA2 with respect to growth regulation is highly conserved among mammals and opens up the possibility of regulating body and organ size in a variety of mammalian species including food and companion animals.

324 FUNCTIONAL ANALYSIS USING SHORT-INTERFERING (si)RNA OF TRAM-6, A NOVEL TRANSCRIPT ASSOCIATED WITH OOCYTES MATURATION IN CATTLE

Maturation of cumulus–oocyte complexes (COCs) with gonadotropins requires transcription of new mRNAs. When COCs are cultured with FSH and the transcriptional inhibitor DRB, cumulus expansion and germinal vesicle breakdown (GVBD) are arrested. Differential mRNA display was used to identify a novel transcript associated with maturation of COCs (TRAM-6) that is expressed during early maturation but not when maturation is inhibited by DRB. The objective of this study was to use siRNAs targeted against TRAM-6 mRNA to assess its functional role in oocyte maturation. Exp. 1: Pools of 60–10 bovine COCs (n e 5/trt) were randomly assigned to culture in maturation medium consisting of 0.5 mL TCM-199 with 2.5 μg FSH, 0.5 μg estradiol, and 10p estrous cow serum with or without DRB (120 μM) or with increasing doses of siTRAM-6 (25, 50, or 100 nM). Exp. 2: COC pools (n e 4/trt) were cultured in maturation medium with one of the following treatments: control, 120 μM DRB, 100 nM non-specific siRNA (siNS), or 100 nM siTRAM-6. After 4 h of culture, COC pools were used to assess levels of mRNA for TRAM-6, VEGF (vascular endothelial growth factor), and GAPD (glyceraldehyde-3-phosphate dehydrogenase) by semiquantitative RT-PCR. Exp. 3: COCs were cultured in maturation medium for either 8 h (n e 9 pools/trt) or 20 h (n e 7 pools/trt) with one of the following treatments: control, 120 µM DRB, 100 nM siNS, or 100 nM siTRAM-6. Cumulus expansion was graded every 4 h. At the end of culture, a subset of COCs from each treatment was used to assess oocyte meiotic stage. Remaining COCs were used to assess TRAM-6, VEGF, and GAPD mRNA. Data were analyzed using ANOVA and Duncans test. At 4 h of culture, relative expression of TRAM-6:GAPD mRNA (least squares means ± SEM) was decreased in the DRB and the 100 nM siTRAM-6 treatments but was unaffected by 25 nM TRAM-6, 50 nM siTRAM-6, or 100 nM siNS (Exp. 1: 100 ± 13pa, 17 ± 13pb, 101 ± 13pa, 60 ± 13pab and 48 ± 13pb for control, DRB, 25 nM siTRAM-6, 50 nM siTRAM-6, and 100 nM siTRAM-6, respectively; abcP < 0.05; Exp. 2: 100 ± 10pa, 14 ± 10pb, 106 ± 10pa and 68 ± 10pc for control, DRB, 100 nM siNS, and 100 nM siTRAM-6, respectively; abcP < 0.05). There was no effect of treatment on expression of VEGF and GAPD mRNA, both of which are present in COCs at the start of culture and are unrelated to TRAM-6 mRNA. At 8 h, GVBD was inhibited by treatment with DRB and siTRAM-6 (68 ± 8pa, 3 ± 8pb, 72 ± 8pa, and 30 ± 8pc for control, DRB, siNSs and siTRAM-6, respectively; abcP < 0.05). At 20 h, the incidence of metaphase II (MII) oocytes was decreased in the DRB and siTRAM-6 groups (96 ± 8pa, 9 ± 8pb, 94 ± 8pa, and 56 ± 8pc for control, DRB, siNS, and siTRAM-6, respectively; abcP < 0.05). Cumulus expansion was inhibited (P < 0.05) by DRB but was not affected by any other treatment. In summary, TRAM-6 siRNA decreased the expression of TRAM-6 mRNA in bovine COCs at 4 h of culture as well as the proportions of oocytes in GVBD at 8 h and in MII at 20 h of culture, but did not affect cumulus expansion. In conclusion, these data are consistent with the identification of a novel mRNA transcript, TRAM-6, that has a functional role in regulating meiotic maturation in bovine COCs. This work was supported by USDA Grant #2002–35205–12810.