Vectors for production of growth hormone
The present invention provides novel compositions to transfect cells for production of growth hormone (GH). These novel compositions also are used to produce germline transgenic birds that can successfully pass the transgene encoding growth hormone to their offspring. These novel compositions include components of vectors such as a vector backbone, a novel promoter, and a gene of interest that encodes for GH, and the vectors comprising these components. In one embodiment these vectors are transposon-based vectors. The present invention also provides methods of making these compositions and methods of using these compositions for the production of GH in vitro and in vivo. In one embodiment the GH is human (h)GH.
1. A vector comprising:
a modified transposase gene operably linked to a first promoter, wherein the nucleotide sequence 3′ to the first promoter comprises a modified Kozak sequence, and wherein a plurality of the first twenty codons of the transposase gene are modified from the wild-type sequence by changing the nucleotide at the third base position of the codon to an adenine or thymine without modifying the amino acid encoded by the codon;
a multiple cloning site capable of receiving a gene of interest;
transposon insertion sequences recognized by a transposase encoded by the modified transposase gene, wherein the transposon insertion sequences flank the multiple cloning site; and,
one or more insulator elements located between the transposon insertion sequences and the multiple cloning site, wherein the one or more insulator elements comprise a lysozyme replicator element, a combination of a lysozyme replicator element and an HS4 element, or a matrix attachment region element, wherein when the gene of interest is inserted into the multiple cloning site, the start codon of the gene of interest is located no more than 3006 bp and no less than 1659 bp from the one or more insulator elements located 5′ to the start codon of the gene of interest.
2. The vector of claim 1 , further comprising a second promoter, wherein the second promoter is SEQ ID NO: 14 or SEQ ID NO: 15.
3. The vector of claim 1 , further comprising a gene encoding for growth hormone inserted into the multiple cloning site.
4. The vector of claim 3 , wherein the vector comprises SEQ ID NO: 31 or SEQ ID NO:32.
5. A transposon-based vector comprising:
a modified transposase gene operably linked to a first promoter, wherein the nucleotide sequence 3′ to the first promoter comprises a modified Kozak sequence, and wherein a plurality of the first twenty codons of the transposase gene are modified from the wild-type sequence by changing the nucleotide at the third base position of the codon to an adenine or thymine without modifying the amino acid encoded by the codon;
one or more genes of interest encoding growth hormone operably-linked to one or more additional promoters, wherein the one or more genes of interest encoding growth hormone and their operably-linked promoters are flanked by transposon insertion sequences recognized by a transposase encoded by the modified transposase gene; and,
one or more insulator elements located between the transposon insertion sequences and the one or more genes of interest encoding growth hormone, wherein the start codon of the gene of interest is located about 3006 bp to about 1659 bp from the one or more insulator elements located 5′ to the start codon of the gene of interest, and wherein the one or more insulator elements comprise a lysozyme replicator element, a combination of a lysozyme replicator element and an HS4 element, or a matrix attachment region element.
6. A method of producing growth hormone comprising:
transfecting a cell with a vector comprising
a modified transposase gene operably linked to a first promoter, wherein the nucleotide sequence 3′ to the first promoter comprises a modified Kozak sequence, and wherein a plurality of the first twenty codons of the transposase gene are modified from the wild-type sequence by changing the nucleotide at the third base position of the codon to an adenine or thymine without modifying the amino acid encoded by the codon;
one or more genes of interest encoding growth hormone operably-linked to one or more additional promoters, wherein the one or more genes of interest encoding growth hormone and their operably-linked promoters are flanked by transposon insertion sequences recognized by a transposase encoded by the modified transposase gene; and,
one or more insulator elements located between the transposon insertion sequences and the one or more genes of interest encoding growth hormone, wherein the start codon of the gene of interest is located about 3006 by to about 1659 by from the one or more insulator elements located 5′ to the start codon of the gene of interest, and wherein the one or more insulator elements comprise a lysozyme replicator element, a combination of a lysozyme replicator element and an HS4 element, or a matrix attachment region element;
culturing the transfected cell in culture medium;
permitting the cell to release growth hormone into the culture medium;
collecting the culture medium; and,
isolating the growth hormone.
7. The method of claim 6 wherein the vector comprises SEQ ID NO:31 or SEQ ID NO:32.
8. The method of claim 6 wherein the growth hormone is human growth hormone.