1/11/2024 0 Comments Macvector use primer3![]() Therefore, late functions of Tc-svb in epidermal and appendage differentiation are strongly affected in Tc-svb CRISPR mutant embryos, while the segmentation phenotype is milder that Tc-svb-RNAi knockdown due to maternal contribution of Tc-svb (Ray et al., in preparation). In addition to segmentation defects and transformation toward thoracic identity, other phenotypes observed in Tc-svb CRISPR mutants include incipient spiracles (possibly a secondary effect of cuticle thinning leading to a defect in the development of tracheal rings) sensory bristles that are shorter and thicker leg segment boundaries that are not clearly defined missing leg bristles unsclerotized pretarsi with soft, rounded apices and antennae lacking the terminal setae. Gene disruption leads to mRNA truncation after the insertion site, since Tc-svb expression is absent in homozygous mutants. The mutagenic cassette is inserted into exon 2, that is within the open reading frame upstream of the region encoding the DNA-binding zinc finger domain. ( H) Magnified ventral view of a svb knockdown showing transformed abdominal segments bearing legs (red asterisks) and fused abdominal segments. One or more of these ectopic legs are often reduced to stumps. Irrespective of body size and phenotypic strength, segments with extra legs are always A1 and A2 (red asterisks). Sometimes urogomphi ( u) are present ( G). ![]() Leg segments get progressively shorter and more rounded, and the pretarsi reduced. ( E–G) Stronger phenotypes are more antero-posteriorly shortened and the last abdominal segment and the telson may either be absent or fused with the anterior segments beyond distinction. Thoracic legs display shorter leg segments (See Figure 1). While most of the abdominal segments are often present, segment boundaries are less distinct especially in the latter abdominal segments where they may be fused, and telson appendages are usually missing. ( A–D) In larvae with weaker segmentation phenotypes, bodies are shortened along the anterior-posterior axis. Source data for Figure 1G are found in Source Data File 1. Data were analyzed by one-way ANOVA using multiple comparison tests against wild-type values. ( G) Quantification of the reduction in leg length, estimated by the distance between coxa/trochanter boundary to the pretarsus tip. The scheme represents a larval leg with corresponding segments pictures portray an example of prothoracic leg ( T1) in wildtype, mlpt, Tc-ubr3 and Tc-svb inactivation. ( F) Knockdown of each of the three genes leads to shortened ‘true-thoracic’ legs, with rounded and often poorly separated distal segments. ![]() Depletion of mlpt, Tc-svb, and Tc-ubr3 causes highly similar segmentation phenotypes, characterized by a reduction in segment number, the presence of extra-legs (arrows) suggestive of transformation of abdominal segments towards a thoracic fate (red asterisks), and the frequent absence of terminal structures. Cuticle phenotypes of Tribolium first instar larvae from following genotypes: wild type ( A), mlpt RNAi ( B), Tc-ubr3 RNAi ( C), Tc-svb RNAi ( D), and Tc-svb CRISPR mutant ( E).
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