Floxed Stop Codon
Floxed Stop Codon - Related Videos
10:41
Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers
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Cited by 5 •
2019
This study presents an alternative strategy to the conventional toxic analog-based method in identifying amino acid overproducers by using rare-codon-rich markers to achieve accuracy, sensitivity, and high-throughput simultaneously.
08:48
Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice
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Cited by 13 •
2022
The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.
10:35
Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis
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Cited by 12 •
2020
Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.
09:37
Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells
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2021
Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.
02:28
Leaky Scanning
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2020
During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA. Marilyn Kozak discovered that the sequence RCCAUGG (where R stands for...
FAQs
Cre recombinase recognizes the two loxP sites surrounding the stop sequence and promotes site-specific recombination. This removes the intervening stop cassette, leaving the downstream gene positioned for transcription from its promoter. The key outcome is a switch from transcriptional blockage to gene activation, provided the construct and promoter are arranged to support expression after excision.
Flanking the stop sequence with loxP sites makes that cassette the defined target for Cre-mediated recombination. The arrangement allows the blocking element to be removed rather than merely bypassed or suppressed. Consequently, gene activation depends on the intended recombination event, giving the engineered system a specific genetic control point for downstream expression.
Activation depends primarily on where and when Cre recombinase is present. Cre distribution can restrict recombination to selected tissues or engineered cell populations, while its timing or induction condition can determine when the stop cassette is removed. The promoter controlling the downstream gene also matters because it governs transcription once the cassette no longer blocks the construct.
A floxed stop codon separates the presence of a transgene from its immediate expression. Before Cre-mediated excision, the stop cassette blocks transcription; after the relevant recombination event, the downstream gene can be expressed under promoter control. This conditional arrangement lets researchers associate genetic effects with selected tissues, time points, or treatment conditions rather than activating the gene universally.
A typical design places the transcriptional stop cassette between loxP sites upstream of the selected downstream gene and under the control of an appropriate promoter. The engineered cell or organism is then combined with a context in which Cre is present in the desired location or condition. Comparing Cre-exposed and non-Cre contexts reveals the conditional behavior of the construct.
In lineage tracing, conditional activation can mark cells in which the relevant Cre-mediated recombination occurs. Because the stop cassette initially prevents downstream expression, the engineered construct provides a genetic record linked to the selected Cre context. This helps researchers follow descendants or analyze cell populations associated with a particular tissue, developmental setting, or experimental condition.
They allow a transgene’s effects to be examined in selected tissues or under defined treatment conditions instead of across the entire engineered system. Researchers can therefore isolate the relationship between gene activation and a specific biological context. In bioengineering, this supports experimental models designed to test function while limiting interpretation to the cells or conditions in which activation was intended.