HIPP11 Antibody

What is the HIPP11 (H11) locus and why is it important for transgene integration?

The HIPP11 (H11) locus is an intergenic region located between the Eif4enif1 and Drg1 genes on mouse chromosome 11. It functions as a genomic "safe harbor" locus that allows predictable integration of transgenes without disrupting endogenous gene function. This locus is particularly valuable because it supports high-level gene expression and exhibits higher rates of recombination compared to other common integration sites like Rosa26 . The H11 locus doesn't have an endogenous promoter, making it especially suitable for conditional expression of transgenes driven by tissue-specific promoters or inducible expression systems .

For researchers working with antibody expression systems or creating humanized mouse models, the H11 locus provides a reliable integration site that maintains stable transgene expression across generations without affecting mouse viability or fertility .

How does the H11 locus compare to other genomic safe harbor sites?

The H11 locus offers several advantages over other commonly used safe harbor sites:

• Higher recombination rates compared to the Rosa26 locus, resulting in improved targeting efficiency

• Greater level of transgene expression in vivo in mice

• Supports stable transgene expression in embryonic stem cells and their differentiated derivatives

• Allows biallelic expression of targeting cassettes

• Does not have an endogenous promoter, providing flexibility for various promoter systems

In human cells, the H11 locus (located on chromosome 22) is considered preferable to commonly used loci such as AAVS1, ROSA26, and HPRT1 for stable transgene expression, particularly in embryonic stem cells and their derivatives . For mouse models, the H11 locus has demonstrated advantages over the widely used Rosa26 locus due to its higher rate of mitotic recombination and robust support for global gene expression from promoters like pCA .

What methods can be used to detect successful integration at the H11 locus?

Successful integration at the H11 locus can be verified through several complementary methods:

• PCR verification: Design primers that span the integration junction (one primer outside the homology arm and one within the integrated construct) to confirm site-specific integration .

• Western blot analysis: For protein-expressing constructs, antibodies against the expressed protein or against tags (such as HA-tag for TIR1 or GFP for reporter constructs) can be used to confirm expression .

• Sequencing: Confirm the exact insertion site and integrity of the integrated sequence through Sanger or next-generation sequencing.

• Functional assays: For constructs like rtTA or Cre recombinase, functionality can be confirmed by crossing with reporter lines (such as TetO-H2BGFP or Ai14) and observing expected patterns of reporter expression .

In the study by Chen et al., PCR primers were designed outside the homologous arms to exclude detection of random insertions, ensuring only genomic DNA with correct insertion produced the desired PCR products .

How can the H11 locus be utilized for the auxin-inducible degron (AID) system in stem cell research?

The H11 locus provides an excellent integration site for the TIR1 transgene, which is essential for the auxin-inducible degron (AID) protein degradation system. This approach offers several methodological advantages:

• Integration of the TIR1 expression cassette at the H11 locus ensures stable expression during stem cell differentiation, which is crucial when studying proteins in rapidly changing environments like proliferation and differentiation .

• The method enables rapid and reversible degradation of target proteins upon addition of auxin, allowing temporal control of protein function without genetic deletion.

• For implementation, researchers can use CRISPR-Cas9 to target the H11 locus with a donor vector containing HA-tagged TIR1 sequence flanked by homology arms specific to the H11 locus.

• Both constitutive and conditional (Cre-dependent) TIR1 expression systems can be established at the H11 locus.

This system has been successfully used to target stable proteins like components of the SMC5/6 complex, achieving rapid protein degradation within hours compared to the two days required with conventional Cre-mediated knockout approaches .

What are the considerations for designing homology arms for CRISPR-Cas9 mediated integration at the H11 locus?

Designing effective homology arms is critical for successful CRISPR-Cas9 mediated integration at the H11 locus:

• Homology arm length: Studies have used homology arms of approximately 700bp for mouse H11 targeting (689bp for 5' arm and 720bp for 3' arm) .

• Proximity to cut site: Position the homology arms close to the cut site. In one study, the 5' homology arm was designed to span 3bp away from the cut site and the 3' homology arm 5bp away from the cut site .

• Specificity: Ensure homology arms are specific to the H11 locus to prevent off-target integration.

• PCR amplification strategy: Homology arms can be amplified by PCR using genomic DNA from the target species (e.g., C57BL/6 mouse) as template .

• Incorporation method: Homology arms can be incorporated into donor vectors using methods like Gibson assembly to create the final targeting construct .

For validation of correct integration, design PCR primers that anneal outside the homology arms to ensure detection of only correctly targeted insertions and exclude random integration events .

How efficient is CRISPR-Cas9 mediated integration at the H11 locus compared to other methods?

CRISPR-Cas9 mediated homology-directed repair (HDR) at the H11 locus has demonstrated remarkably high efficiency:

• Studies report integration efficiencies of up to 36% for large transgenes at the H11 locus in mice. In one study, 8 out of 22 pups (36%) harbored the correct albumin-rtTA insertion after microinjection and embryo transfer .

• The H11 locus has been shown to facilitate highly efficient integration of large transgenes, such as the human CD1A promoter and coding region, via CRISPR-Cas9 mediated HDR .

• The higher rate of mitotic recombination at the H11 locus compared to Rosa26 suggests better accessibility to site-specific recombinases like φC31 integrase .

The efficiency of H11 targeting makes it particularly valuable for generating transgenic models, as it reduces the screening burden and accelerates the development of new mouse lines. This high efficiency is likely due to the open chromatin structure and accessibility of the H11 locus .

How can the H11 locus be used for liver-specific transgene expression systems?

The H11 locus can be effectively utilized for liver-specific expression by incorporating tissue-specific promoters. A methodological approach includes:

• Design a construct where a liver-specific promoter (such as the albumin promoter) drives the expression of a desired transgene or system component like rtTA.

• Create a donor vector containing the tissue-specific cassette flanked by H11 homology arms.

• Use CRISPR-Cas9 to target the H11 locus and introduce the construct via homology-directed repair.

Chen et al. demonstrated this approach by generating H11-albumin-rtTA transgenic mice, where the albumin promoter drives liver-specific expression of rtTA. When crossed with TetO-H2BGFP mice, H2BGFP expression was observed exclusively in the liver upon doxycycline induction, confirming both tissue specificity and inducibility .

This system can be further expanded by crossing H11-albumin-rtTA mice with TetO-Cre and Ai14 reporter mice to generate triple transgenic mice with liver-specific, doxycycline-inducible Cre-mediated recombination capabilities .

What is the stability of transgene expression from the H11 locus during stem cell differentiation?

The stability of transgene expression from the H11 locus during stem cell differentiation is a critical consideration for developmental studies:

• The H11 locus supports stable transgene expression in both human and mouse embryonic stem cells (ESCs) and their differentiated derivatives .

• This stability makes the H11 locus particularly valuable for studying protein functions during differentiation processes, where expression must be maintained as cells transition between states.

• When using systems like the auxin-inducible degron (AID), integration of the TIR1 component at the H11 locus ensures the degradation system remains functional during differentiation .

• The locus appears to reside in a region of chromatin that remains accessible during development, unlike some other integration sites that may become silenced during specific differentiation pathways.

For researchers developing antibody expression systems or studying protein function during development, the stable expression characteristics of the H11 locus provide a significant advantage over loci that may be subject to developmental silencing .

What are the recommended protocols for validating H11 locus targeting in transgenic models?

A comprehensive validation protocol for H11 locus targeting should include:

• Genomic PCR screening:

  • Design primers that span the 5' and 3' integration junctions

  • Include primers outside the homology arms to ensure detection of only correctly targeted events

  • Verify PCR products by sequencing to confirm precise integration

• Expression validation:

  • For protein-coding transgenes: Western blot using antibodies against the expressed protein or epitope tags

  • For fluorescent reporters: Direct fluorescence microscopy or flow cytometry

  • For inducible systems: Test functionality by administering inducer (e.g., doxycycline for rtTA systems)

• Functional verification:

  • For tissue-specific systems: Confirm expression is limited to target tissues

  • For inducible systems: Verify both activation upon inducer administration and silencing after inducer withdrawal

  • For recombinase systems: Cross with reporter lines to validate recombination activity

• Germline transmission testing:

  • Confirm stable inheritance of the targeted allele in subsequent generations

  • Verify continued functionality in offspring

Chen et al. demonstrated these principles by using PCR with primers designed outside the homology arms, followed by Western blot analysis using GFP antibody to confirm doxycycline-induced expression of H2BGFP in the liver of their H11-albumin-rtTA/TetO-H2BGFP double transgenic mice .

How can the H11 locus be used for conditional expression systems?

The H11 locus is particularly well-suited for conditional expression systems due to its lack of an endogenous promoter. Implementation methodologies include:

• Tet-On/Off systems:

  • Integration of rtTA or tTA at the H11 locus, driven by constitutive or tissue-specific promoters

  • Crossing with TetO-regulated transgenic lines to achieve inducible expression

  • Administration of doxycycline to control expression timing

• Cre-Lox conditional systems:

  • Integration of a floxed-STOP cassette between the promoter and transgene at the H11 locus

  • Expression only occurs in cells expressing Cre recombinase

  • Can be combined with inducible Cre systems for temporal control

• Auxin-inducible degron system:

  • Integration of TIR1 at the H11 locus (constitutive or conditional)

  • Tagging target proteins with AID

  • Addition of auxin induces rapid protein degradation

For example, Pryzhkova and Jordan created a conditional TIR1 expression vector by incorporating a floxed STOP cassette between the EF1α promoter and TIR1 sequence in their H11 targeting vector. This allows TIR1 expression only after Cre-mediated excision of the STOP cassette .

Similarly, Chen et al. demonstrated the utility of the H11 locus for liver-specific, doxycycline-inducible expression using the albumin promoter to drive rtTA expression .

What strategies can overcome low integration efficiency at the H11 locus?

While the H11 locus generally allows high integration efficiency, researchers may occasionally encounter challenges. Several optimization strategies can help:

• CRISPR guide RNA design:

  • Test multiple sgRNAs targeting the H11 locus

  • Select guides with high on-target and low off-target scores

  • Consider testing guides in cell culture before proceeding to animal models

• Homology arm optimization:

  • Adjust homology arm length (typically 500-1000bp works well)

  • Ensure homology arms are positioned close to the cut site

  • Verify that homology arms are free of polymorphisms relative to the target genome

• Donor template format:

  • Compare circular vs. linear donor templates

  • For large inserts, consider using BACs or reducing the insert size

• CRISPR delivery method:

  • For zygotes: Optimize microinjection parameters or try electroporation

  • For ES cells: Test different transfection reagents or nucleofection protocols

• Selection strategies:

  • Include a selection marker in the donor template when possible

  • Consider using positive-negative selection approaches

The high natural recombination rate at the H11 locus compared to other sites like Rosa26 contributes to its targeting efficiency, but these optimization steps can further improve success rates .

How can researchers detect and troubleshoot unwanted recombination or rearrangements at the H11 locus?

Detecting and troubleshooting unwanted recombination events requires systematic screening:

• Comprehensive PCR-based screening:

  • Design multiple primer pairs spanning the entire integration site

  • Include primers that would detect head-to-tail or head-to-head concatemers

  • Use long-range PCR to detect large-scale rearrangements

• Southern blot analysis:

  • Design probes targeting the integrated sequence and flanking genomic regions

  • Use multiple restriction enzymes to generate distinct banding patterns

  • Compare observed vs. expected fragment sizes to detect rearrangements

• Next-generation sequencing approaches:

  • Targeted sequencing of the H11 locus and integrated construct

  • Whole genome sequencing to detect potential translocations

  • Long-read sequencing (PacBio, Nanopore) to resolve complex structural variations

• Functional verification:

  • Test expression levels of the integrated transgene

  • Compare expression across different founder lines

  • Check for unexpected expression patterns that might indicate position effects

If unwanted recombination is detected, it's advisable to generate and screen additional founder lines rather than proceeding with problematic integrations that may compromise experimental results or complicate data interpretation.