...mice can be included in breeding strategies identical to those used for breeding heterozygous mice. In ...all of the offspring in the F1 generation, your breeding scheme should start with parental mice that are...detect your targeting vector or gene of interest. 2. Breed your parental mice to generate F1 Once you’ve performed...locus (e.g. Cre) and heterozygous at the other. 3. Breed your F1 generation Now, your goal is to generate...as progeny from future generations. 4. Continue breeding your colony You’ll want to generate enough mice...controls in your experiment - use mice that were bred in your colony, as opposed to ordering control mice...harbor similar epigenetic modifications. Special breeding cases Whether your mouse colony consists of genetically...

...When it comes to breast cancer, Hoshinho and colleagues found that after injecting breast cancer cells into... Xing et al., 2010) revealed precisely that for breast cancer. What are tumor microenvironments made ...proliferation. However, the lack of caveolin 1 in breast cancer also induces changes of stromal cells in...characterization of the tumor microenvironment in breast cancer." Cancer cell 6.1 (2004): 17-32. PubMed ...Myoepithelial cells: autocrine and paracrine suppressors of breast cancer progression." Journal of mammary gland biology...Diana, and Fernando Schmitt. "Microenvironment in breast tumorigenesis: Friend or foe?." Histology and histopathology...tumor recurrence and poor clinical outcome in human breast cancers." The American journal of pathology 174.6...

...yet? If not, just remember that the next holiday break is just a few weeks away. Is your favorite science...

... 30125270. Changing the way we detect viral outbreaks Article contributed by Izzy Mueller  Listen to...highly contagious, it is essential to contain outbreaks when they surface. But detection of the virus ... podcast segment The human proteome contains a breadth of modular domains that facilitate protein-protein...

... DNA lesion results from breakage of both DNA strands - a double-strand break (DSB). DSBs can be caused...double-strand break repair (DSBR), synthesis-dependent strand-annealing (SDSA), and break-induced repair... homology directed repair fix DNA double-strand breaks?  In eukaryotic cells, repair of DSBs occurs primarily...have these central steps: The 5’ DNA end of the break is resected by nucleases to create a ssDNA 3’ overhang...covered in detail here. Classical double-strand break repair (DSBR) In the classical DSBR pathway, the...ligated to fill the gap, thus completing SDSA.   Break-induced repair (BIR) pathway BIR is characterized... it has biological importance for the repair of breaks that lack a second end to enable DSBR or SDSA. ...

...under one of two broad classifications: inbred or outbred. Inbred strains share a common genetic background...of different genome editing techniques. Inbred and outbred strains Mouse strains used in research can...manipulation. Common inbred strains include C57BL/6, Balb/c, and 129. Conversely, outbred strains were originally... genetically diverse gene pool. Outbred strains are strategically bred to maintain maximum genetic heterozygosity...helps improve the reproducibility of experiments. Inbred mice are particularly useful for understanding ...the genetic diversity of the human population. Outbred strains of mice are useful for comparing dominant...not useful for genetic engineering. Examples of outbred strains include Swiss Webster and Harlan (Hsd) ...

...PMC5171807. 6. Machleidt, T., et al. (2015) NanoBRET-A novel BRET platform for the analysis of protein-protein...known as bioluminescent resonance energy transfer (BRET). The fluor then emits light at a tissue-penetrating...infection of host cells. The fluorescent moiety of the BRET reporter was used to sort successfully infected/...substrate for NanoLuc luciferase. Both GpNLuc and OgNLuc BRET reporters produced bright signals without exogenous...emission near 600 nm – a clear demonstration of why a BRET reporter can improve deep tissue imaging. The constructs...Chu, J., et al. (4), likewise, wanted to produce a BRET probe for imaging. These authors set out to design...Enhanced Nano-Lanterns to other cellular proteins. BRET-based biosensors utilizing NanoLuc® luciferase Many...

...Cas9-induced breaks by NHEJ As illustrated below, NHEJ-mediated repair of Cas9-generated breaks is useful...exogenous DNA fragment at a Cas9 targeted break (or pair of breaks) by NHEJ-dependent repair (“pop-in” insertion...to generate a targeted single strand break; when two such breaks are introduced near each other, in opposite...be easily programmed to make a DNA double strand break (DSB) in the genome wherever the user chooses. After...synthesis. NHEJ also finishes repair of most types of breaks in tens of minutes – an order of magnitude faster...principal means by which CRISPR/Cas9-introduced breaks are repaired. The following factors are required...compatible DNA ends together, thus repairing the break. This is a simplified, streamlined version of this...

...by committing a break to performing HR before other factors were loaded onto the break, potentially shuttling...cells already have for repairing DNA double strand breaks – (HR). HR is the dominant homology-directed repair... often mutated in cancer cells, specifically in breast and ovarian lines commonly used in lab. It is recommended...1: Early steps in repair of a DNA double strand break by homologous recombination.    Designing donors...knock-in location. HDR depends on resection at the break site to form ssDNA and uses the ends of the ssDNA... observed when inserts are within 10 bps of the break, making the cut off essentially as part of the templated...promote HDR by generating ssDNA overhangs at the break site – an early HR step (Moreno-Mateos, et al., ...

...as Bioluminescence Resonance Energy Transfer (BRET). BRET is similar to Förster Resonance Energy Transfer...acceptor. TRUPATH uses an updated version of BRET called BRET2. Figure 1: TRUPATH measures heterotrimeric...resulting in decreased BRET2 signal. With increased concentration of drug, the BRET2 signal decreases. ...excite an acceptor fluorophore. The difference for BRET is that the energy transfer is between a luminescent...bioluminescence resonance energy transfer 2, or BRET2. With binding of the drug, the alpha and beta subunits... The TRUPATH BRET2 biosensor system was developed by constructing 14 luciferase donor Rluc8-Gα chimeras...emission. The activation state can be measured by a BRET2 ratio, which is the ratio of GFP2 emission to RLuc8...

...locus. The first two breaks are induced via a generic PITCh-gRNA; the third break by an insertion locus-specific.... But have you heard of another double-stranded break repair mechanism: MMEJ (microhomology-mediated end-joining...methods for repairing DNA after a double-stranded break. HDR copies the sequence from a repair template ... regions of homology flanking a double-stranded break yield precise deletion of the sequence between the...flanking the GFP-Puro cassette. Three double stranded breaks are necessary for knock-in: one on either side ...locus-specific gRNA. These double stranded breaks allow for two sets of microhomologies (5’ and 3’) to anneal...cleaves genomic DNA. These three double stranded breaks are repaired by two rounds of MMEJ. This double...

...also set aside Breakout Rooms to divide the audience into small sessions and do a quick break to chat. This...participants to group together. Breakout Rooms can be used as a break, but can also be used to brainstorm...that showing something different from time to time breaks up the monologue a little and gives participants... even the termination of a project.” Take brain breaks Meetings can get very long sometimes, and interaction...longer we are on it – so it is important to take breaks. After 1 hour (or another designated time) of meeting...raise their hands or signal the approach of a brain break, where everyone stands up, disappears from the video...

...which causes the breaks) and host cell DNA repair factors (which repair those breaks) makes Cas9 extremely...thus, causes two types of editing outcomes: Cas9 breaks repaired by the non-homologous end-joining (NHEJ...gene sequences (thus inactivating genes), while breaks repaired by homology directed repair (HDR) pathways...Using substrate DNA labeled on both sides of the break, we determined that Cas9 held tightly to three of...length, With 36bp on the PAM-distal side of the break and 91bp on the PAM-proximal side, consistently...repair of the gene would be disastrous (i.e. if breaking the gene is worse than leaving the mutation alone...

...derivatives. Unmodified Cas9 causes double stranded DNA breaks that commonly result in a complete loss of function...genes as they are subject to an increase in dsDNA breaks that can cause cell death. Thus, CRISPR inhibition...library! Now also available as a lentiviral prep. Calabrese: human CRISPRa sgRNA library In addition to inhibiting... A and B. Sanson et al. demonstrated that the Calabrese library substantially identified more hits than...al., 2015). Root and Doench also compared the Calabrese library to screens performed with open reading...for resistance to MEK inhibition. Although both Calabrese and the ORF overexpression library identified ...in complement to study gene function. Find the Calabrese pooled library and lentiviral prep here! Applications...

... while FokI then induces a double strand break. These breaks can then be repaired by homologous recombination...discovery of the Cas proteins in 2005 was a major breakthrough in the world of genetic engineering as CRISPR-Cas...just like its predecessor induce a double-strand break at the target genomic location. This is typically...and CRISPR-Cas) is that they induce double-strand breaks (DSBs) in the genome. These technologies rely on...affected by CRISPR.”  Prime editing The latest breakthrough in genome editing is prime editing, which was...Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576:149–157 . https://doi....

...double-strand breaks (DSBs) evaluated by sequencing (GUIDE-seq) (Tsai et al. 2015) and direct in situ breaks labeling...sequence all DSBs, may miss unpredicted off-target breaks Prediction algorithms: A good place to start...that may still lead to off-target double-stranded breaks (Tsai et al. 2015). Additionally, current algorithms...2013). GUIDE-seq and BLESS detect double-stranded breaks and do not require high sequencing read counts .... "Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases." Nature biotechnology...et al. "Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing." Nature methods...

...found my outline, read over Figure 4, took a deep breath, and, re-oriented, continued presenting. The whole... a memory lapse, I simply pause and take a deep breath before the panic sets in. Most of the time, that...let's quickly go over it now'? Maybe take a deep breath?" Once you answer the question, you need only to... If you're also nervous, try taking a few deep breaths. Then, identify the specific things making you ...t go perfectly,” can help. (And do remember to breathe!)   Public speaking as a transferable skill The...other job you may choose to pursue. So take a deep breath–you got this!  Additional resources on the Addgene...

... (ABEs) chemically modify target bases without breaking the DNA backbone, making them efficient and precise...nickase (nCas9) to avoid introducing double-strand breaks. The only things base editors really need from ...also called apurinic or apyrimidinic sites and abbreviated as AP sites. To explore this further, the team...that is known for sometimes causing double-strand breaks (DSBs). The Fei-Long Meng Lab did in-depth profiling...strand is excluded as ssDNA. DSB Double-Stranded Break A cut to both strands of DNA. Indel Insertion/... (2024). C-to-G editing generates double-strand breaks causing deletion, transversion and translocation...

...negatively-charged membranes (Davis et al., 2002). Polybrene is one such cationic reagent that is commonly ...efficiency by 10-fold (Davis et al., 2002). However, polybrene is highly sensitive to freeze-thaw cycles and ...be stored in single-use aliquots. Never use a polybrene solution that has been thawed more than once. ...in cells that are sensitive to the toxicity of polybrene (e.g., hematopoietic or primary cells), and has...., Jeffrey R. Morgan, and Martin L. Yarmush. "Polybrene increases retrovirus gene transfer efficiency ...

...1970s when Sydney Brenner brought it into the lab to study neuronal development (Brenner, 1973). C. elegans...scientific research. Yes, the same kind we use in breads and other baked goods! Yeast is cheap, simple and...maintained as they are housed in large groups, easily bred, and produce 50-300 eggs at a time. Zebrafish embryos...developed for emerging model organisms! References Brenner, Sydney. "The genetics of Caenorhabditis elegans...