Integration, stability and expression of the E. coli phytase transgene in the Cassie line of Yorkshire Enviropig™

The genomic structure and generational stability of the transgene carried by the Cassie (CA) line of the transgenic Enviropig™, a prospective food animal, are reported here. This transgene is composed of the Escherichia coli phytase coding sequence regulated by the mouse parotid secretory protein promoter to direct secretion of phytase in the saliva. In the CA line the transgene integrated in chromosome 4 is present as a concatemer of three copies, two in a head to tail orientation and the third in a reverse orientation 3′ to the other copies with a 6 kbp deletion in the 5′ promoter region. The overall size of the integrated transgene complex is 46 kbp. During integration a 66 kbp segment of the chromosome was deleted, but a BLAST search of the segment from a GenBank clone did not reveal any essential genes. The transgene integration site was stable through 9 generations analyzed. Phytase activity in the saliva was similar among 11 day old hemizygous boars and gilts and remained relatively constant through nine generations of hemizygous pigs. However, as the pigs grew there generally was a gradual decrease in activity that stabilized when pigs reached the finisher phase of growth (4–6 months old). Homozygous pigs exhibited 1.5 fold higher phytase activity (P < 0.0001) than that of hemizygous littermates. Moreover, no differential salivary phytase activity was seen in hemizygotes arising from CA-Yorkshire and CA-Duroc breed outcrosses, suggesting that expression of the transgene is unaffected by genetic background. This data demonstrates that an exogenous phytase gene can be stably transmitted and expressed in the salivary glands of a domestic food animal.

[1]  P Green,et al.  Base-calling of automated sequencer traces using phred. II. Error probabilities. , 1998, Genome research.

[2]  R. Palmiter,et al.  Production of transgenic rabbits, sheep and pigs by microinjection , 1985, Nature.

[3]  C. Centeno,et al.  Phytase and acid phosphatase activities in plant feedstuffs. , 2000, Journal of agricultural and food chemistry.

[4]  Henry A. Erlich,et al.  PCR Technology: Principles and Applications for DNA Amplification , 1989 .

[5]  M. Ribaudo,et al.  Trends and Developments in Hog Manure Management: 1998-2009 , 2012 .

[6]  C. Forsberg,et al.  Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities. , 1999, Canadian journal of microbiology.

[7]  P. D’Eustachio,et al.  Cellular DNA rearrangements and early developmental arrest caused by DNA insertion in transgenic mouse embryos , 1987, Molecular and cellular biology.

[8]  E. Wagner,et al.  The human beta-globin gene and a functional viral thymidine kinase gene in developing mice. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Bishop Jo.  Chromosomal insertion of foreign DNA. , 1996 .

[10]  P. Gerhardt,et al.  Methods for general and molecular bacteriology , 1994 .

[11]  H. Würtele,et al.  Illegitimate DNA integration in mammalian cells , 2003, Gene Therapy.

[12]  A. Low,et al.  Current Concepts of Digestion and Absorption in Pigs , 1980 .

[13]  J. Phillips,et al.  Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control , 2001, Nature Biotechnology.

[14]  R. Rohan,et al.  Direct sequencing of PCR-amplified junction fragments from tandemly repeated transgenes. , 1990, Nucleic acids research.

[15]  S. Carpenter.  Phosphorus control is critical to mitigating eutrophication , 2008, Proceedings of the National Academy of Sciences.

[16]  R. Schinazi,et al.  Retrovirus Reverse Transcriptases Containing a Modified YXDD Motif , 2005, Antiviral chemistry & chemotherapy.

[17]  David Garrick,et al.  Repeat-induced gene silencing in mammals , 1998, Nature Genetics.

[18]  Y. Sakaki,et al.  Mechanism of chromosomal integration of transgenes in microinjected mouse eggs: sequence analysis of genome-transgene and transgene-transgene junctions at two loci. , 1993, Gene.

[19]  Cross Reactivity.  ANTIGEN-antibody reactions. , 1952, The American journal of medicine.

[20]  J. Bishop,et al.  Chromosomal insertion of foreign DNA. , 1996, Reproduction, nutrition, development.

[21]  R. Higuchi,et al.  Simple and Rapid Preparation of Samples for PCR , 1989 .

[22]  R. Palmiter,et al.  Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Cecil W. Forsberg,et al.  Pigs expressing salivary phytase produce low-phosphorus manure , 2001, Nature Biotechnology.

[24]  P. K. Smith,et al.  Measurement of protein using bicinchoninic acid. , 1985, Analytical biochemistry.

[25]  C. Forsberg,et al.  Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities. , 2000, Canadian journal of microbiology.

[26]  R. Petters,et al.  Phenotypic stability of Pro347Leu rhodopsin transgenic pigs as indicated by photoreceptor cell degeneration , 2011, Transgenic Research.

[27]  H. Steinfeld,et al.  Issues and options in addressing the environmental consequences of livestock sector's growth. , 2010, Meat science.

[28]  J. Lam,et al.  Antigen-Antibody Reactions , 2007 .

[29]  P. Green,et al.  Base-calling of automated sequencer traces using phred. I. Accuracy assessment. , 1998, Genome research.