FC26-06 - Development of interneurons expressing serotonin 3A subtype receptor and sleep regulation

IntroductionSerotonin (5-HT) is known to play a key role in a number of psychiatric disorders. The effects of abnormal 5-HT neurotransmission on the modulation of circadian rhythm have been proposed to be one of the mechanisms underlying these disorders.Objectives and aimsHere we describe developmental characteristics of interneurones (IN) expressing 5-HT type 3A receptor (5-HT3AR) in mice. This receptor is a ligand-gated ion channel which activation leads to a rapid excitatory response in neurons.The expression of 5-HT3AR during embryonic development in mice is mainly restricted to caudal ganglionic eminence (CGE) and entopedoncular area (EPA). We hypothesized that these regions are those specifically involved in the neurogenesis of the IN expressing 5-HT3AR.MethodsTo assess this hypothesis, we used homochronic in utero grafts. Cells were collected from CGE and EPA regions of transgenic mice embryos expressing Green Fluorescent Protein under the control of 5-HT3AR gene promoter. After in utero transplantation in non transgenic embryo, we examined at adult age the location of the grafted cells, as well as their morphological properties.ResultsWe observed that CGE-derived cells gave rise to IN that mostly populated the neocortex and the hippocampus, whereas EPA-derived cells integrated the amygdala, the pyriform cortex and the Ventro-Lateral Pre-Optic (VLPO) nucleus. Moreover, the VLPO EPA-derived cells are similar to “sleep active cells” found in this region and responsible of sleep induction.ConclusionThese results lead to the better understanding of the IN expressing 5HT3AR development. Its activation is possibly implicated in triggering sleep mechanismes.

2 NEW IVF TRANSGENESIS STRATEGY IN BOVINE USING CELL CYCLE INHIBITORS AND MOSAICISM REVERSION BY CLONING

All the techniques available for transgenic animal production are inefficient. A new transgenic embryo production strategy was developed, consisting on injection of oolema vesicles coincubated with DNA into fertilized embryos. To improve this technique, we evaluated the effects of 1) linear and circular covalently closed plasmid structures; 2) cell cycle inhibitors (DMAP and Dehydroleucodine, DhL) during first pronuclear phase. The effect of the transgene on DNA double strand breaks (DSBs) was also tested. Transgenic blastomeres produced by these strategies were cloned to reverse mosaicism. To this aim, COCs were IVM and subjected to IVF (Bracket and Oliphant, 1975). Then, presumptive zygotes were injected with oolema vesicles incubated with plasmid (OVIP) or with plasmid alone. The plasmid employed was linearized or circular pCX-EGFP. Two treatments were evaluated: 2 mM DMAP (OVIP+DMAP) or 1 uM DhL (OVIP+DhL) for 6 h (15 to 21 h post IVF). Dynamics of egfp expression were daily evaluated. The DNA DSBs were measured by immunocytochemistry against phosphorylated H2AXγ histone. Cloning of IVF transgenic blastomeres was included for the groups OVIP+DMAP, OVIP and for plasmid alone. Rates of egfp expression were higher for linear than circular pCX-EGFP [58/81(71%), 45/68(66%), 51/84(60%), 34/63(53%) v. 24/72(33%), 30/72(41%), 31/93(33%), 18/53(33%), for OVIP+DMAP, OVIP+DhL, OVIP, and plasmid alone respectively; P < 0.05]. As well, egfp expression was delayed after circular plasmids injection (at day 3 post fertilization, a maximum of 7% for circular and over 40% for linear injected groups). For the groups injected with circular pCX-EGFP, there were not differences in egfp blastocysts rates [16/26(61%); 11/23(47%); 13/28(46%), and 5/11(45%) for OVIP+DMAP, OVIP+DhL, OVIP, and plasmid alone respectively]. Except for the group injected with plasmid alone, linear plasmid structure improved egfp blastocysts rates [21/22(95%); 17/22(77%); 22/26(84%) and 10/19(52%) for OVIP+DMAP, OVIP+DhL, OVIP, and Plasmid alone, respectively]. In all cases, DMAP incubation tended to improve egfp rates. Preliminar confocal images showed higher H2AXγ foci number after OVIP injection previous to DMAP or DhL than for DMAP or DhL alone (mean foci number: 122, 72, and 135 for OVIP+DhL, OVIP+DMAP and OVIP v. 38 and 49 for IVF+DhL or +DMAP). Homogenous egfp expression was detected in up to 22% of embryos used as donors for cloning. All cloned blastocysts derived from OVIP+DMAP or OVIP were transgenic (total n = 10). For plasmid alone injection, 20% (1/5) blastocysts lost egfp expression after cloning. Homogenous expression was detected in 100% of OVIP and OVIP+DMAP cloned embryos (33/33 and 40/40), statistically different to plasmid alone (32/42; 76%). In conclusion, the best conditions for IVF mediated transgenesis were OVIP injection, using linearized plasmid structures, followed by DMAP. The presence of the transgene was related to an increase in DNA DSBs. Cloning of egfp blastomeres resulted in mosaicism reversion. This new IVF mediated transgenesis technique represents an interesting alternative for transgenic animal production.

4 MYOSTATIN GENE KNOCKDOWN THROUGH LENTIVIRAL VECTOR-MEDIATED DELIVERY OF shRNA FOR IN VITRO PRODUCTION OF TRANSGENIC BOVINE EMBRYOS

The main goal of husbandry and beef cattle production is to enhance performance rates, for example, weight gain. Myostatin is referred to as a negative regulator of skeletal muscle growth. Genetic engineering of this character in order to produce double muscling animals that can transmit to future progeny will enhance its usefulness. The present research aimed to analyze myostatin inhibition through lentiviral-mediated delivery of shRNA in mouse myoblast culture and the feasibility of the lentiviral-mediated delivery of shRNA into in vitro-produced transgenic bovine embryos. In order to achieve knockdown of myostatin in cell and embryo culture, a lentiviral vector was constructed with ubiquitin C promoter-driven GFP gene (green fluorescent protein) and shRNA to suppress myostatin gene expression driven by the U6 promoter. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR of myostatin and GAPDH genes. Later, bovine oocytes were in vitro-matured and the lentiviral vector was microinjected into the oocyte perivitelline space (2.5 � 106 IU mL-1) after mechanical and chemical cumulus cell removal. Non-microinjected mature oocytes were considered as control. After microinjection, oocytes were fertilized and cultured in vitro. After 4 and 9 days of culture, embryos were evaluated by epifluorescence microscopy. The GFP-positive embryos were green under fluorescence. Cell morphology and embryo development rate data were analyzed by Minitab Release 14 Statistical Software (Minitab, Inc., State College, PA, USA), submitted to ANOVA, and compared by Tukey test (P d 0.05). Real-time PCR data were analyzed by Pair-Wise Fixed Reallocation Randomization Test using REST2005 software. Cell morphology results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells as the transducted group progressed less to myotubes than in the control group. A lower amount of myostatin mRNA after 72 h of differentiation indicated an inhibition tendency by real-time PCR. In relation to the transgenic embryo production, 96.9 � 0.34% (62.65) developed to cleavage, 80.24 � 4.38% (51/65) were GFP-positive, and 50.95 � 3.37% (26/65) achieved blastocyst stage. After hatching, 3.07% (2/65) of GFP-positive embryos maintained fluorescence. In relation to the control group, the cleavage rate was 93.81 � 0.68% (61/65); the blastocyst rate 38.34 � 2.36% (25/65), and none were fluorescent. In conclusion, myostatin gene knockdown was effectively performed by lentiviral vector-mediated delivery of shRNA. Thus, novel studies about the efficiency of this vector on transgenic embryo production can be performed. This work was supported financially by FAPESP 03/0156-9.

Overexpression of the glycolytic enzyme pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) in developing transgenic tobacco seed results in alterations in the onset and extent of storage lipid deposition

Microscopy and fatty acid analyses were used to characterize the storage lipids in transgenic Nicotiana tabacum L. (tobacco) ovules demonstrating constitutive expression of a gene encoding pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) from the protist Giardia lamblia. Transmission electron microscopy demonstrated significant differences in the size and number of lipid droplets in endosperm and embryo proper cells, regardless of the presence of the transgene; confocal fluorescence microscopy confirmed constitutive differences in lipid droplets of the endosperm and embryo tissues at maturity. Microscopy of developing tissues, however, showed that lipid droplets were deposited up to 48 h sooner in the transgenic embryo proper while deposition of endosperm lipids was unchanged. Cell counts of sectioned tissues confirmed that transgenic embryo growth was enhanced. Fatty acid analyses showed that fatty acid methyl ester (FAME) concentrations of stearic (18:0), linoleic (18:2), and linolenic (18:3) fatty acids were significantly higher in the larger, transgenic embryos, while those of palmitic (16:0) and oleic (18:1) were lower, suggesting that overexpression of PFP is responsible for altering both growth and development of young ovules. At maturity, palmitic and stearic FAMEs were significantly lower, but no other morphological or biochemical differences between the control and transgenic seeds could be measured.Key words: triacylglycerols, Nicotiana tabacum, transgenic, embryo development, storage lipids.

178 RESISTANCE TO CODLING MOTH: EXPRESSION OF SYNTHETIC CRYIA(C) GENES IN TRANSGENIC WALNUT EMBRYOS

Insecticidal crystal protein fragments (ICPFs) of Bacillus thuringiensis (Bt) encoded by cryIA(c) gene were shown in diet incorporation studies to be lethal to codling moth (CM; Cydia pomonella) the key insect pest for walnut. However transformed walnut tissues expressing cryIA(c) with Bt codon usage patterns and native DNA sequence revealed very low levels of expression in planta. To correct this problem synthetic versions of one of these genes, cryIA(c) was used to transform walnut tissue. A total of 61 individual transgenic embryo lines were obtained. 34% of these lines (21/61) were high expressors (“class A”) demonstrating 80 to 100% mortality of first in star CM larvae and displaying no further larval development. Twelve clones (20%) were designated “class B” and these showed a marked retardation of larval development and a mortality between 40 to 79%. Embryos from the remaining 28 lines designated “class C” (46%). although transformed, were indistinguishable from the control (untransformed embryos) and showed a mortality of 0 to 39%.