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Expression of threonine-biosynthetic genes in mammalian cells and transgenic mice

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Abstract

Threonine is a nutritionally essential amino acid (EAA) for the growth and development of humans and other nonruminant animals and must be provided in diets to sustain life. The aim of this study was to synthesize threonine in mammalian cells through transgenic techniques. To achieve this goal, we combined the genes involved in bacterial threonine biosynthesis pathways into a single open reading frame separated by self-cleaving peptides (2A) and then linked it into a transposon system (piggyBac). The plasmids pEF1a-IRES-GFP-E2F-his and pEF1a-IRES-GFP-M2F-his expressed Escherichia coli homoserine kinase and threonine synthase efficiently in mouse cells and enabled cells to synthesize threonine from homoserine. This biosynthetic pathway occurred with a low level of efficiency in transgenic mice. Three transgenic mice were identified by Southern blot from 72 newborn mice, raising the possibility that a high level of expression of these genes in mouse embryos might be lethal. The results indicated that it is feasible to synthesize threonine in animal cells using genetic engineering technology. Further work is required to improve the efficiency of this method for introducing genes into mammals. We propose that the transgenic technology provides a promising means to enhance the synthesis of nutritionally EAAs in farm animals and to eliminate or reduce supplementation of these nutrients in diets for livestock, poultry and fish.

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Abbreviations

EAA:

Nutritionally essential amino acid

HPLC:

High-performance liquid chromatography

KHB:

Krebs–Henseleit bicarbonate buffer

PCR:

Polymerase chain reaction

RT-PCR:

Reverse transcription polymerase chain reaction

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Acknowledgments

We thank Dr. Sen Wu for providing us with the pEF1a-PB plasmid and materials for cell culture. This study was supported by the National Transgenic Breeding Project of China (grant 2013ZX08006-004), Chinese Universities Scientific Funds (2012RC024), and the Thousand-People Talent program at China Agricultural University.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China

    Yurui Zhang, Ran Zhang, Yunping Dai & Ning Li

  2. State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, 100193, China

    Zhaolai Dai & Guoyao Wu

  3. Department of Animal Science, Texas A and M University, College station, TX, 77843, USA

    Guoyao Wu

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  1. Yurui Zhang
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  2. Zhaolai Dai
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  3. Guoyao Wu
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  4. Ran Zhang
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  5. Yunping Dai
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  6. Ning Li
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Correspondence to Ning Li.

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Zhang, Y., Dai, Z., Wu, G. et al. Expression of threonine-biosynthetic genes in mammalian cells and transgenic mice. Amino Acids 46, 2177–2188 (2014). https://doi.org/10.1007/s00726-014-1769-0

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