MM - Breed Me Deep Sir.mp4
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What can I say, we have a serious weakness for a tight small pumpkin shaped ass! It laterally makes us weak in the knees. Mac has a perfect pump-kin butt, truth be told, we invited him over for a solo stroke video and it quickly turned into more (cock sucking ass eating and facials, all on MMDirects) Butt as soon as we saw that pumpkin we knew we had to have him back for a full on fuck flick. When we see a sweet little pumpkin butt like Macs, it makes us crazy. As soon as we met Mack for the first video, we realized he was going to be fun, he is so sexy cute, has a great sense of humor and he was so comfortable with us and then when we saw that PHAT ASS we fell in lust lol when the video was over I put my camera down and jumped in the shower with Hunter, and much to our surprise Mack picked up the camera when we were out of the room and recorded us a little message telling us how much fun he had, so sexy and so sweet lol. You will love this deep ass fuck session
We present a simple CRISPR-Cas9 method to generate zebrafish F0 knockouts suitable for studying behaviour and other continuous traits. The protocol uses a set of three synthetic gRNAs per gene, combining multi-locus targeting with high mutagenesis at each locus. The method consistently converts > 90% of injected embryos into biallelic knockouts that show fully penetrant pigmentation phenotypes and near complete absence of wild-type alleles in deep sequencing data. In parallel, we developed a quick and cheap PCR-based tool to validate gRNAs whatever the nature of the mutant alleles. The F0 knockout protocol is easily adapted to generate biallelic mutations in up to three genes in individual animals. The populations of F0 knockout animals generated by the method are suitable for quantitative analysis of complex phenotypes, as demonstrated by mutation of a circadian clock component and by meticulous replication of multi-parameter behavioural phenotypes of a genetic model of epilepsy.
(A) The headloop score (HL score) for a given sample (here, tyr locus A larva 1) was calculated as the ratio between the headloop PCR band intensity (HL) and the standard PCR band intensity (std). As the standard PCR band intensity represents the sum of wild-type and mutated alleles, and the headloop PCR band intensity represents the mutated alleles only, the ratio approximates the proportion of mutated alleles in the sample. (B) Percentage of mutated reads (deep sequencing) as a function of headloop score. Each data point corresponds to one targeted locus in one F0 knockout animal. Some samples were artificially created to simulate mediocre gRNAs by mixing genomic DNA from an injected embryo with genomic DNA from the uninjected control in a 1:1 (diluted , squares) or 1:3 ratio (diluted , triangles). For these, the percentage of mutated reads was not measured by deep sequencing but estimated by dividing the percentage of mutated reads of the original sample by 2 (diluted ) or 4 (diluted ). The dark grey dashed line is the line of best fit by linear regression: proportion of mutated reads = 0.33 + 0.69 headloop score; R2 = 0.44. Headloop score is a significant predictor of the proportion of mutated reads, p
In the first round, each gRNA set is injected followed by deep sequencing or headloop PCR to confirm mutagenesis, thereby controlling the false negative rate of a screen. Headloop PCR is cheap, robust, and requires only a single step, which makes it easily adapted to high-throughput screening. No specialist equipment is required, as opposed to qPCR (Yu et al., 2014), high resolution melting analysis (HRMA) (Samarut et al., 2016), or fluorescent PCR (Carrington et al., 2015). Unlike deep sequencing, qPCR, and HRMA, it is also flexible with respect to the size of amplicons and so is sensitive to a wide range of alleles, from small indels to large deletions between targeted loci. It can be used to assay the efficiency of any gRNA, with no restrictions on target sequence that might be imposed by the use of restriction fragment length polymorphism (Jao et al., 2013), for example. The products of headloop PCR are also compatible with different sequencing methods, should further analysis of mutant haplotypes be required.
It is precisely because discovering novel phenotypes is the main challenge of F0 knockout methods that an essential criterion for success was achieving high phenotypic penetrance and close to complete removal of wild-type alleles for test genes. When studying phenotypes that vary continuously in the population, such as locomotor activity (Figure 6) or circadian clock period (Figure 5B), it is not possible to readily identify animals in the F0 knockout population that display a phenotype, in contrast to situations in which animals exhibit overt morphological differences. Therefore, the majority of the animals in the F0 knockout population need to be bona fide knockouts. Continuous traits often have the added challenge that they are not phenotypes whose spatial variation is evident in individual animals (Watson et al., 2020), in contrast to developmental phenotypes like formation of electrical synapses (Shah et al., 2015) or distribution of microglia (Kuil et al., 2019). Therefore, most cells in each F0 knockout animal must carry biallelic loss-of-function mutations. With the pigmentation phenotypes golden (slc24a5 knockout) and sandy (tyr knockout), and the deep sequencing data at 32 targeted sites, we demonstrate that the F0 knockouts generated by the present method show very little mosaicism, in the sense that most wild-type alleles are removed, and hence can be used to discover novel phenotypes that require most or all the cells of most F0 knockout animals to be mutated.
In the case of the small indels around each targeted site, we discovered through deep sequencing that there was substantial diversity in alleles, including within individual animals. Most mutations at each locus are present at low frequencies in individual animals (> 95% mutations detected at each locus are present at frequencies below 10% in individual animals, Author response image 1). This may be because the mutation happened later in development or in a cell that did not contribute many daughter cells. High coverage is necessary to detect and quantify this diversity. Many of these alleles would likely be missed by the lower coverage of an amplification-free experiment.
To answer specifically the first question raised, the order when targeting gradually more loci (Figure 1E,F) followed the IDT ranking when selecting a single crRNA per exon (see Material and methods, section RNP pooling). Therefore, when targeting a single locus, only the top predicted gRNA is injected; when targeting two loci, the top two predicted gRNAs are injected together; etc. Based on the deep sequencing data (Figure 2A), this amounts to essentially the same as picking the order randomly within the top 3 or 4 gRNAs, as the top predicted gRNA of the set is only rarely the most mutagenic one in practice. Indeed, when we sequenced 10 genes targeted by three or four-gRNA sets, there were only 3 sets in which the top predicted gRNA by IDT (gRNA A) was also the top performing gRNA (slc24a5, tbx5a, scn1lab). This is what is expected by chance alone (if the most mutagenic gRNA of each set was drawn randomly, 31% of gRNA sets would have the top predicted gRNA as most mutagenic gRNA). In other words, it is not possible to guess above chance the IDT ranking from the mutagenesis levels alone. 59ce067264
https://www.samposd.com/forum/ilban/plants-vs-zombies-battle-for-neighborville-fre
Porsche Design looks again at 50 years of advancement
The brand is usually celebrating its anniversary which has a series of limited-edition products. high quality replica watches
Even in 2022, few brands are because involved in as many categories since Porsche Design - via watches and sunglasses in order to electronics and now even brand name homes, non-e do it in addition to Porsche Design. The lifestyle manufacturer, which first emerged being a spinoff of the original Porsche brand, is celebrating their 50th anniversary and release a series of limited-edition products to be able to mark this important motorola milestone mobiel phone.
While the celebration obviously provided the Porsche Design team with the best opportunity to delve into its records, reaching such a milestone likewise brought back memories of the past. Naturally, the luxury industry has created a lot since Porsche Design first found its legs in 1972.
“I think the concept of luxury, no less than in the Western world, has changed a lot, ” Roland Heiler, Chief Design Officer of the Porsche Design Lifestyle Group and Taking care of Director of Studio FA Porsche, told Elite Visitor. “Luxury is no longer about currently being ostentatious, conspicuous and excellent rich. In fact , I think it includes returned to being far more understated and subtle, with an increase of and more people appreciating premium quality (over the past 50 years). ” replica luxury Watches
Luckily for Porsche Design, this idea of understated extravagance has been ingrained into the brand’s ethos from the beginning. “For us all, it hasn’t changed and so dramatically because we’ve by no means been in the luxury space, ” Heiler adds.
Rather, Porsche Design fans have always been, as Heiler applies it, “people who we appreciate you high quality and like to individual something for a long time. ” Throw-away items and fashion trends don't have any place here.
Having this mentality from the beginning has served Porsche Design well in today’s world, exactly where sustainability is increasingly greatly regarded. As with everything the brand can, while eco-consciousness is top rated of mind, it’s worn out a subtle way.
“In our targeted group, people expect durability, but they don’t want it on the outside of the product, ” affirms Jan Becker, CEO from the Porsche Lifestyle Group. “The products have to be sustainable, nevertheless [our customers] don’t expect us for you to hype it or placed it in ads or claim it explicitly. It’s mare like a subtle expectation. ”
“Some of the [Porsche Design] items on the market are 50 years outdated, like the Black Chronograph one particular and our famous P’8478 sunglasses, which have sold spanning a million pairs since 1978, ” he added. “Our tools are lifelong companions, which is typically the embodiment of sustainability as well as directly related to Porsche Design. ” Jacob & Co. Epic X Tourbillon Baguette
To showcase the stamina of not only the actual goods, but also the appeal of it has the timeless designs, which are nonetheless as fresh today while they were in 1972, Porsche Layout has dug up several of its most iconic patterns from its vast assortment and re-released them.
The anniversary edition includes two models: Is a fully restored Porsche 1972 911 Targa, which will be auctioned later this year. According to Heiler, the entire anniversary project started off with this restoration.
“We wanted to figure out how to read the car as if it had been developed by F. A. Porsche 39 years ago, ” he says, “when he had just given up the career as a car custom made to become a product designer and put up his new business. ”
To the interior, for example , Heller and his men delved into the archives to discover the perfect fabric. “We planned to be true to the times, ” he says. “We reviewed [the car’s] history with Porsche Classic and found a wool textile with a small check… that had been used in a car designed for a close relative in the Sixties. ” best replica watches
The fabric is just like the houndstooth-style Porsche Pepita used from 1963 to help 1973, making this remastered auto a product of its time period, but with the performance of any modern car.
From this specially restored 911 comes the new 2022 model. Limited to 750 units, often the 911 50th Anniversary Porsche Design Edition is based on the actual 911 Targa 4 GTS, but with a few key specifics that harken back to the actual brand’s origins.
Like its restored brother, the 2022 model characteristics an archival check pic and updated manufacturing techniques to ensure it meets modern-day standards. There are some special splashes on the exterior, too, most notably some sort of commemorative plaque with FA Porsche’s signature. “It’s entertaining, ” Heiler says.
Porsche Design has reached into the watch racks to re-release an renowned product to celebrate its 50th anniversary. Available only to those that purchase the car, the Time counter 1 - 911 Edition 50Y Porsche Design can be a modern interpretation of the initial Chronograph 1, aka Porsche Design’s first watch.
“The rebirth in the Chronograph 1 is a very significant element for our company, ” says Heiler. “It’s wonderful to bring this original view, which was founded in 1972, back in the market after so many decades without it. ” replica Richard Mille McLaren
While goal was to stay faithful to the original, naturally some alterations were made, including an up-to-date movement and replacing stainlesss steel with titanium in the case and also bracelet.
“From a design perspective, many of us stayed very close to the authentic, ” Heiler explains. “We chose titanium because, in addition to gold, all our circumstances are titanium. As a firm, we think it’s the best stuff to make watches… I think buyers will like that because is considered nice to see that the layout hasn’t become dated, when everything else has improved. ”
For hobbyists who missed out, nonetheless Porsche Design has released yet another watch - the Chronograph 1 - All Black Numbered Edition, which will be offered this November and can be pre-ordered now. Like the other everlasting nature watches, the All African american Numbered Edition largely maintains the original’s matte african american dial and all-important reddish colored seconds hand, while up to date elements, like the current Porsche Design logo, solidify the status as a 21st-century enjoy.
Although the celebration is a celebration of the prior 50 years, Porsche Design, often committed to innovation, is already planning to the future. “For the a centesimal anniversary, we have a lot to take into consideration in the coming years, ” Becker says. “We ought to think about how to spread each of our brand. ”
One of the upcoming plans is usually to enter the hotel market, along with at least 15 properties supposed to be built in partnership together with Steigenberger Hotels. “I feel this is one of the biggest milestones intended for Porsche Design, ” Likas? said. “We are currently seeking our first location… It is going to be a sensation. ” replica Bell & Ross BR 05