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Results for “H11 locus”

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PubMed2025

Validation of caprine H11 and the Rosa26 platform for transgene integration via CRISPR-based system: investigations on stable transgene expression and genetic biosafety.

CRISPR/Cas9 technology is an efficient tool for site-specific livestock gene editing. However, to minimize potential disruption of host genome function, exogenous genes should be integrated into well-characterized genomic loci, such as H11 or Rosa26, which have been empirically validated for stable transgene expression. This study established a multi-dimensional assessment system to evaluate the biological applicability of the H11 locus and the widely used Rosa26 targeting platform as sites for targeted integration of exogenous genes in goats. Donor cells carrying the enhanced green fluorescent protein (EGFP) reporter gene at the H11 and Rosa26 loci were generated via CRISPR/Cas9-mediated homology-directed repair; this was followed by somatic cell nuclear transfer to produce transgenic cloned embryos and healthy offspring. Multi-dimensional analyses revealed the following. At the cellular level, there was stable and efficient EGFP expression at integration sites, with donor cells maintaining normal cell cycle progression, proliferation capacity, and apoptosis levels, and with no alterations in the transcriptional integrity of adjacent genes. At the embryonic level, there was sustained EGFP expression across pre-implantation embryonic stages, with developmental metrics statistically indistinguishable from wild-type embryos. Finally, at the individual level, cloned offspring exhibited growth phenotypes consistent with wild-type counterparts, and EGFP showed broad-spectrum expression in eight tissues. This study establishes the first CRISPR/Cas9-based crossscale (cellular-embryonic-individual) validation in goats, demonstrating that the H11 and Rosa26 loci support efficient and stable transgene integration in goats. These results provide a precise and predictable technical framework for livestock genetic improvement.

Animals↗

PubMed1997

Selective loss of the hepatic phenotype due to the absence of a transcriptional activation pathway.

Liver-enriched trans-acting factors hepatocyte nuclear factor-1 alpha (HNF1 alpha) and -4 (HNF4) are components of a transcriptional activation pathway that is thought to play a major role in hepatic gene activation. We previously described the isolation and characterization of distinct classes of hepatoma variants which lack the HNF4-->HNF1 alpha pathway (1). In order to determine the influence of the HNF4-->HNF1 alpha pathway on hepatic gene expression, genetic rescue experiments were done using hepatoma variant line H11 as a model system. Results suggest that this pathway is required for basal expression of a number of endogenous hepatocyte-specific genes. Complementation groups were established by fusion of H11 cells with other variant lines. Lastly, introduction of human chromosome 20 (containing the HNF4 locus) or randomly-marked human chromosomes into H11 cells failed to rescue the hepatic phenotype, suggesting that what appears to be a 'simple' defect may involve multiple genetic loci.

Animals↗

PubMed1998

Rescue of the HNF4 --> HNF1alpha pathway in hepatoma variant cells containing human chromosome 12.

Expression of liver-enriched trans-acting hepatocyte nuclear factors 1alpha (HNF1alpha) and 4 (HNF4) is correlated with the hepatic phenotype in cultured rat hepatoma cells. We have used a hepatoma variant cell line, H11, that specifically lacks the HNF4 --> HNF1alpha pathway as a model to understand mechanisms controlling hepatic gene expression. We have introduced randomly marked human chromosomes into H11 cells and have isolated a number of microcell hybrids that have rescued hepatic gene expression, including HNF4, HNF1alpha, and alpha1-antitrypsin. Chromosomal analysis of cell hybrids showed that the rescued hepatic phenotype correlated closely with the presence of human chromosome 12p sequences. Although the gene encoding HNF1alpha is located on chromosome 12q24, its retention was not required to rescue the hepatic phenotype. Thus, we suggest that a locus on human chromosome 12p plays an important role in maintenance of hepatic gene expression through activation of the HNF4 --> HNF1alpha pathway.

Animals↗