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Characterization of Expression in Mice of a Transgene Containing 3.3 kb of the Human Lactase-Phlorizin Hydrolase (LPH) 5′ Flanking Sequence

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Abstract

Background and Aim

The regulation of human intestinal lactase-phlorizin hydrolase remains incompletely understood. One kb of pig and 2 kb of rat 5′-flanking sequence controls correct tissue, cell, topographic, and villus LCT expression. To gain insight into human LCT expression, transgenic mouse lines were generated from 3.3 kb of human LPH 5′ flanking sequence from a lactase persistent individual fused to a human growth hormone (hGH) reporter bounded by an insulator.

Methods

Four lines were identified in which reporter expression was specifically detectable in the intestine and no other organ, two of which demonstrated hGH expression specific to small and large intestine. Quantitative RT-PCR was carried out on proximal to distal segments of small intestine at fetal days 16.5 and 18.5 and at birth, postnatal days 7 and 28 in line 22.

Results

In fetal intestine, hGH expression demonstrated a proximal to distal gradient similar to that in native intestine. There was no significant difference between hGH expression levels at 7 and 28 days in segment 3, the midpoint of the small intestine, where expression of endogenous lactase is maximal at 7 days and declines significantly by 28 days. Distal small intestine displayed high levels of hGH expression in enteroendocrine cells, which were shown to be a subset of the PYY cells.

Conclusions

Thus, a 3.3-kb LPH 5′ flanking sequence construct from a lactase persistent individual is able to maintain postnatal expression in transgenic mice post weaning.

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Acknowledgments

The authors are indebted to Stuart Orkin, MD, for generous provision of the insulator, Andrew B. Leiter, PhD for provision of the PYY antibody, and to Christina Piaseckyj for superb technical support. Transgenic mice were generated in the Center of Excellence for Molecular Hematology supported by a grant from the NIDDK (DK49216) to Stuart H. Orkin. Supported in part by NIH MERIT Award DK R37 32658, NIH Research Grant R01 DK 061382, and the Harvard Digestive Disease Center P30 DK 34854.

Conflict of interest

None of the authors has a conflict of interest of any kind.

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Authors and Affiliations

  1. Gastrointestinal Cell, Molecular Biology Laboratory, Division of Gastroenterology and Nutrition, Children’s Hospital Boston, Enders 609.2, 300 Longwood Avenue, Boston, MA, 02115, USA

    Nana Yaa Baffour-Awuah, Eveline Delemarre, Jarom Heijmans, David Boerwinkel, Selma Algra, John J. Fialkovich, Shabana Islam, Tjalling Bosse, Richard J. Grand, Stephen D. Krasinski & Robert K. Montgomery

  2. Division of Hematology, Department of Medicine, Children’s Hospital Boston, Boston, MA, USA

    Yuko Fujiwara

  3. Harvard Digestive Disease Center, Children’s Hospital Boston, Boston, MA, USA

    Richard J. Grand, Stephen D. Krasinski & Robert K. Montgomery

  4. Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Richard J. Grand, Stephen D. Krasinski & Robert K. Montgomery

Authors
  1. Nana Yaa Baffour-Awuah
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  2. Eveline Delemarre
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  3. Yuko Fujiwara
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  4. Jarom Heijmans
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  5. David Boerwinkel
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  6. Selma Algra
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  7. John J. Fialkovich
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  8. Shabana Islam
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  9. Tjalling Bosse
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  10. Richard J. Grand
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  11. Stephen D. Krasinski
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  12. Robert K. Montgomery
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Corresponding author

Correspondence to Robert K. Montgomery.

Additional information

Nana Yaa Baffour-Awuah and Eveline Delemarre contributed equally to this research.

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Supplemental figure 1

. Representation of the 3.3-kb human LPH construct used in this study (see Methods for details). Supplemental figure 2. The transgene does not significantly affect the weight of line 22 mice. Transgenic mice were weighed weekly and compared to their hGH-negative litter mates. Weight is plotted in grams with standard deviations shown for wt male (purple), L22 male (red) and L22 female (green). (PDF 77 kb)

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Baffour-Awuah, N.Y., Delemarre, E., Fujiwara, Y. et al. Characterization of Expression in Mice of a Transgene Containing 3.3 kb of the Human Lactase-Phlorizin Hydrolase (LPH) 5′ Flanking Sequence. Dig Dis Sci 56, 59–69 (2011). https://doi.org/10.1007/s10620-010-1480-2

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  • DOI: https://doi.org/10.1007/s10620-010-1480-2

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