top of page

delisasimpsonyoga Group

Public·11 members

Gsx Level 2 Serial Key

I've just purchased GSX and GSX level 2.Every time I try and put the serial number in, nothing happens. It says I'm still in trial mode (and I'm definitely not after spending nearly 40.00).Can someone help?

Gsx Level 2 Serial Key

I have used GSX with Level 2 Expansion successfully for a few years. Since last week's Live Update, when I then start P3Dv4.5,GSX and GSX Level 2 Expansion both open in Trial Mode only. I then have to re-register both programs with my serial numer and they then work as normal. However when I next open P3Dv4.5 both programs open again in Trial Mode.

After completely reinstalling Windows and P3D4.5, I installed GSX + Level2 but mistakenly activated GSX and the Level2 extension with the same serial number from GSX. Everything looked exactly the same - after restarting the simulator, GSX switched to Trial mode.

After that, I carefully examined the purchase orders on Simmarket and found that GSX Level2 has its own serial number. After activation by different numbers separately GSX and Level2 everything worked correctly. So, make sure that you entered TWO different serial numbers when activating.

The multiple sequence alignments that indicate the levels of polymorphismacross this Bf-Gsx regulatory region (Additional file 2: Figure S2), inaddition to helping identify conserved TFBs, also identified highly conservedregions outside of Bf-Gsx-Up1 that could potentially also contribute toexpression. This region of conservation also extends to a second species ofamphioxus, Branchiostoma belcheri [61, 62] (Additional file 2: Figure S2). The 11 B. floridae Gsx-UpProximal sequences and single B. belcheri Gsx-UpProximal sequence were aligned and visualised using VISTA [63] toidentify regions of high/low polymorphism across the regulatory region(Additional file 2: Figure S2). As expected, the Gsx-Up1 region was highlyconserved across B. floridae individuals, but this conservation also extended into the 3' half ofGsx-Up2, perhaps indicating that this sequence may also contribute to thefunction of the region. Interestingly, this same pattern of conservation ismore apparent when comparing between amphioxus species, B.floridae and B. belcheri (Additional file 2: Figure S2). The same is also true for B.lanceolatum (unpublished data). This cross-species analysis also highlights a drop offin sequence conservation at the 3' of the Up1 region, which correlateswith the 3' end of the Up1c minimal enhancer region.

The role of TCF/Lef in the function of the Bf-Gsx-Up1 and Up1c regulatoryregions prompted us to search for further sites that may be contributingtowards the increase in expression efficiency seen in those constructs alsocontaining the 2b region. As the Bf-Gsx-Up1/Up1c regions showed at least somecollaborative effect between TCF/Lef sites, a third site located within theGsx-Up2b region provided a good target for further mutagenesis (Fig. 5a). Byanalysing the effect of mutation on this third site, again as both a singlemutation and in all possible permutations with the existing 'core'TCF/Lef [DELA]1 and [DELA]2 mutations, we aimed to determine if TCF/Lef sitefunction was acting cumulatively and could account for the increase inexpression seen in Bf-Gsx-Up1 + 2b and Up1c-2b, or if this third site couldperhaps buffer against mutations in the 'core' Up1c region,providing a level of redundancy. Thus, the Site[DELA]3 mutation, GTAGGAATTGATGAA was produced (Fig. 5b). Bf-Gsx-Up1 + 2b[DELA]1, containing a mutationof the first 'core' TCF/Lef site, showed a dramatic decrease in CNSexpression overall, though it is most apparent in the nerve cord with sensoryvesicle expression, which decreases from 42.3 % in the wild type Bf-Gsx-Up1 +2b to 10 % in the Bf-Gsx-Up1 + 2b[DELA]1 mutant. Interestingly, theBf-Gsx-Up1 + 2b[DELA]2 mutated construct shows a less significant decrease inexpression in the nerve cord with sensory vesicle expression pattern (23.5%), though nerve cord alone (0.4 %) and sensory vesicle alone (6.7 %) bothshow comparable results to that of the Bf-Gsx-Up1 + 2b[DELA]1 construct (0.2and 4.3 % respectively) (Fig. 5c). These results reveal a disparity in thecontribution to regulatory function between site1 and site2, perhapsexplained by the non-canonical binding sequence of site2, and the ability ofsite3 to compensate for this lower affinity site. However, if both site1 andsite2 are mutated, as in the Bf-Gsx-Up1 + 2b[DELA]1[DELA]2 construct, we seethat nerve cord with sensory vesicle expression decreases dramatically from42.3 % in the WT to 6.5 % in the [DELA]1[DELA]2 mutation (Fig. 5c). This isalso lower than either single core site mutation alone, supporting the ideathat these TCF/Lef sites are functioning cumulatively. However, it is notablethat there is not a complete abolition of expression as in theBf-Gsx-Up1[DELA]1[DELA]2 and Bf-Gsx-Up1c[DELA]1[DELA]2 constructs (Figs. 4c,d and 5c), implying that the third site is able to partially compensate forthe lack of TCF/Lef binding in the Core Up1c region.

When comparing expression in constructs with all TCF/Lef sites mutated, itwas notable that the background head mesenchyme and tail muscle expressioninherent to the pCES construct was different betweenBf-Gsx-Up1c[DELA]1[DELA]2, Bf-Gsx-Up1[DELA]1[DELA]2 and Bf-Gsx-Up1 +2b[DELA]1[DELA]2[DELA]3. As the TCF/Lef mutant constructs became longer, fromBf-Gsx-Up1c[DELA]1[DELA]2 as the smallest to Bf-Gsx-Up1 +2b[DELA]1[DELA]2[DELA]3 as the longest, the pCES background expression alsodecreased. This led to, alongside the lack of CNS expression in all of theseconstructs, high pCES background in Bf-Gsx-Up1c[DELA]1[DELA]2 (52.2 % ofembryos), a small amount of pCES background in Bf-Gsx-Up1[DELA]1[DELA]2 (9.6% of embryos), and a complete abolition of any expression in the longerBf-Gsx-Up1 + 2b[DELA]1[DELA]2[DELA]3 (0 % of embryos) (Fig. 6). Numbers ofembryos displaying background pCES expression within the wild-type constructsare as follows; Bf-Gsx-Up1c, 88.2 % of embryos (255/289), Bf-Gsx-Up1, 45.5 %of embryos (156/343), and Bf-Gsx-Up1 + 2b, 10.9 % of embryos (46/423). Thisthen shows a decrease in the levels of pCES background expression in directresponse to TCF/Lef site mutation, with Bf-Gsx-Up1c >Bf-Gsx-Up1c[DELA]1[DELA]2 (decreasing from 88.2 to 52.2 %), Bf-Gsx-Up1 >Bf-Gsx-Up1[DELA]1[DELA]2 (decreasing from 45.5 to 9.6 %), and Bf-Gsx-Up1 + 2b> Bf-Gsx-Up1 + 2b[DELA]1[DELA]2[DELA]3 (decreasing from 10.9 to 0 %). Itshould be noted that pCES background expression levels in embryos containingthe empty pCES vector (i.e. the reporter with no regulatory elementinsertion) lies at 94.6 % of embryos (142/150), whilst a long butnon-functional region such as Bf-Gsx-Up2 (479 bp) has pCES background within88.8 % of embryos (120/135). This suggests that the decrease in pCESbackground seen in response to increased construct length is also specific tothe Bf-Gsx-Up1 + 2b region. We hypothesise that by removing TCF/Lefactivation, a latent repressive function is unmasked that is spreadthroughout the regulatory region. Thus, in the absence of TCF/Lef binding, asthe region inserted into the pCES multiple cloning site increases in size, itbecomes more able to repress the background activity of the forkheadpromoter. This system, where transcription factor binding is required todrive expression, but absence causes the regulatory element to activelyrepress gene expression, is one that could have widespread implications forgene regulation, allowing a regulatory region to have even greater precisionover the control of the expression of its target gene, only allowingexpression in the presence of a key, primary transcription factor, which inthis case is TCF/Lef.

The ability of the Bf-Gsx-Up regulatory regions to drive LacZ expressionthroughout the CNS of C. intestinalis is intriguing, as these reporters show LacZ expression in homologous tissuesto those expressing Gsx in amphioxus, i.e. the neural tube and the cerebral/sensory vesicle. Thepartial division of function observed in the Bf-Gsx-Up1 + 2b region intonerve cord and sensory vesicle domains may be linked to the two domains ofnative amphioxus expression. In this case, the visceral ganglion and nervecord domain produced by the Bf-Gsx-Up region could correspond to the earlydomain of amphioxus Gsx, expressed at the level of somite 5 [4], whilst the observed sensory vesicledomain might then correspond to the later cerebral vesicle domain ofamphioxus Gsx. The lack of expression in the most anterior sensory/cerebral vesicle regionin both cases supports this and may indicate a defined boundary that ispresent in both the C. intesinalis and amphioxus anterior CNS.

Within the wider chordate phylum, it is possible that there is some conservedregulation of Gsx, as there is the conservation of expression within the anterior CNS,particularly the 'hindbrain' and mid-forebrain as distinct domainsof Gsx expression. In amphioxus, the early domain of Gsx expression is at the level of somite 5, a region thought to have homology tothe vertebrate hindbrain [80, 81]. Also, for vertebrate Gsx genes, thehindbrain domain is also the first to be expressed, as seen in medaka [47], Xenopus [44], and mouse [45, 46]. This indicates that there is possibly a conservedregulatory program within the chordates that leads to the expression of thisinitial hindbrain domain. In addition, the expression of Gsx genes [44]within the mid-forebrain of vertebrates [46, 47], the cerebral vesicle ofamphioxus [4], and the sensory vesicle of C. intestinalis [48] again hints at conserved regulation of Gsx within the chordates. This,in conjunction with the ability of the Bf-Gsx-Up reporter to drive expressionthroughout the CNS of C. intestinalis, suggests that a conserved regulatory pathway may be driving chordate Gsxexpression. 350c69d7ab

  • About

    Welcome to the group! You can connect with other members, ge...

    bottom of page