Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.

Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes.

During fertilization, an egg and a sperm fuse to form a new embryo. Eggs develop from oocytes in a process called meiosis. Meiosis in human oocytes is highly error-prone, and defective eggs are the leading cause of pregnancy loss and several genetic disorders such as Down's syndrome. Which genes safeguard accurate progression through meiosis is largely unclear. Here we develop high-content phenotypic screening methods for the systematic identification of mammalian meiotic genes. We targeted 774 genes by RNA interference within follicle-enclosed mouse oocytes to block protein expression from an early stage of oocyte development onwards. We then analysed the function of several genes simultaneously by high-resolution imaging of chromosomes and microtubules in live oocytes and scored each oocyte quantitatively for 50 phenotypes, generating a comprehensive resource of meiotic gene function. The screen generated an unprecedented annotated data set of meiotic progression in 2,241 mammalian oocytes, which allowed us to analyse systematically which defects are linked to abnormal chromosome segregation during meiosis, identifying progression into anaphase with misaligned chromosomes as well as defects in spindle organization as risk factors. This study demonstrates how high-content screens can be performed in oocytes, and allows systematic studies of meiosis in mammals.

Spire-type actin nucleators cooperate with Formin-2 to drive asymmetric oocyte division.

Oocytes mature into eggs by extruding half of their chromosomes in a small cell termed the polar body. Asymmetric oocyte division is essential for fertility [1], but despite its importance, little is known about its mechanism. In mammals, the meiotic spindle initially forms close to the center of the oocyte. Thus, two steps are required for asymmetric meiotic division: first, asymmetric spindle positioning and second, polar body extrusion. Here, we identify Spire1 and Spire2 as new key factors in asymmetric division of mouse oocytes. Spire proteins are novel types of actin nucleators that drive nucleation of actin filaments with their four WH2 actin-binding domains [2-6]. We show that Spire1 and Spire2 first mediate asymmetric spindle positioning by assembling an actin network that serves as a substrate for spindle movement. Second, they drive polar body extrusion by promoting assembly of the cleavage furrow. Our data suggest that Spire1 and Spire2 cooperate with Formin-2 (Fmn2) to nucleate actin filaments in mouse oocytes and that both types of nucleators act as a functional unit. This study not only reveals how Spire1 and Spire2 drive two critical steps of asymmetric oocyte division, but it also uncovers the first physiological function of Spire-type actin nucleators in vertebrates.

Identification of a novel binding partners for tumor suppressor PTEN by a yeast two-hybrid approach.

AIM: To identify novel PTEN-binding partners. METHODS: The technique of yeast two-hybrid screening was used in this study. A panel of bait constructs was created, containing the C-terminal domain of PTEN, full length PTEN, activated and phosphatase-dead mutants. The expression of LexA-fused baits, their nuclear localization and autoactivation potential were tested according to the standard protocol of Duplex A system. CDNA libraries from Colon Cancer, HeLa and Mouse Embryo were screened with two selected bait constructs. Isolated positive clones were further analysed by mating assay and identified by automated DNA sequencing and database searching. RESULTS: Extensive screening of cDNA libraries with the full length and the C-terminal domain of PTEN led to the identification of 43 positive clones, which were confirmed in mating assay. Sequence analysis indicated that two clones encode AEBP1 (Adipocyte Enhancer Binding Protein 1). CONCLUSION: Our data indicate that the interaction between PTEN and AEBP1 is mediated by their C-terminal and N-terminal domains, respectively. The functional importance of PTEN-AEBP1 interaction is currently under investigation.