MD-2 CRISPR/Cas9 KO Plasmid (h): sc-417315

Overview

LY96 encodes myeloid differentiation factor 2 (MD-2), an extracellular accessory protein that complexes with TLR4 to recognize bacterial lipopolysaccharide and initiate innate immune signaling. MD-2 is required for efficient receptor dimerization and activation of downstream MyD88- and TRIF-dependent pathways, leading to NF-κB and IRF3 signaling and induction of inflammatory cytokines and type I interferon programs. This axis shapes macrophage and dendritic cell responses to Gram-negative bacteria and contributes to immune homeostasis at barrier tissues. Dysregulated TLR4/MD-2 activity has been implicated in chronic inflammatory states and infection-driven pathology, making LY96 a common target for mechanistic studies of pattern recognition receptor signaling.

MD-2 CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the LY96 gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the LY96 locus, alongside the Streptococcus pyogenes Cas9 nuclease, and encodes GFP to enable fluorescent identification and enrichment of successfully transfected cells. This multi-guide strategy increases the likelihood of inducing frameshifts or deletions that produce a functional knockout, offering a more robust alternative to single-guide approaches. DSBs induced at multiple sites are resolved through non-homologous end joining (NHEJ) or, when used with the included HDR donor template, homology-directed repair (HDR) at a defined target site within the locus.

When used in conjunction with the RFP-expressing HDR donor, GFP and RFP fluorescence can be used together to distinguish transfected from edited cell populations, streamlining flow cytometry-based sorting and clone selection workflows.

Homology-Directed Repair (HDR) Donor — Puromycin Cassette with RFP Reporter

For applications requiring confirmed, selectable knockout clones, MD-2 HDR Plasmid (h) includes an HDR donor construct containing a puromycin resistance cassette (PuroR) and a red fluorescent protein (RFP) reporter, flanked by homology arms specific to a defined LY96 target site.
When co-transfected with MD-2 CRISPR/Cas9 KO Plasmid (h):

• The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the LY96 open reading frame.
• RFP fluorescence provides an immediate visual indicator of successful integration, enabling fluorescence-based identification or sorting of edited cells prior to or alongside puromycin selection.
• Successfully edited cells are confirmed through puromycin resistance, substantially reducing clone screening burden.
• This selection strategy is ideal for generating stable, clonal KO cell lines for downstream functional studies, drug screening, or model development.

Cre-lox Cassette Removal System

The HDR donor construct features loxP sites flanking the PuroR-RFP selection cassette to allow clean marker removal following clone confirmation. Transient expression of Cre recombinase via the included Cre Vector: sc-418923 excises the cassette, leaving a minimal residual loxP site within the LY96 locus and eliminating potential confounding effects on downstream assays.
This two-step approach:

• Minimizes disruption to local chromatin architecture and neighboring regulatory elements
• Restores a near-native genomic context at the edited locus
• Enables reuse of the puromycin selection strategy in the same cell line for additional edits

Key Features

• gRNA targeting LY96 exon(s) critical for MD-2 function
• Co-expression of SpCas9 and sgRNA from a single plasmid for simplified delivery
• HDR donor with puromycin resistance for positive clone selection
• loxP-flanked PuroR cassette with Cre recombinase vector for seamless marker removal
• Supplied ready to use for delivery by transfection

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.