Claims for Patent: 5,252,475

Summary for Patent: 5,252,475

Title:	Methods and vectors for selectively cloning exons
Abstract:	The invention relates to a method for selectively cloning exons in which an ukaryotic genomic DNA fragment to be assayed is cloned into a shuttle vector in a cloning site of an intron flanked by exons. Upon expression in eukaryotic cells, exons present in the cloned DNA are spliced to the exons flanking the intron. After amplification of the cDNA of the expressed mRNA with primers specific for the exons in the vector by PCR, selectively cloned exons can be detected due to the size of the PCR product. The invention also relates to vectors used to carry out the method of the invention as well as to exon libraries.
Inventor(s):	Reth; Michael (Freiburg, DE)
Assignee:	Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.V. (Gottingen, DE)
Application Number:	07/725,608
Patent Claims:	1. A method for selectively cloning exons comprising the following steps: (a) cloning a genomic DNA fragment to be assayed for exons into a cloning site of a vector having the following features: (aa) said vector is a shuttle vector for prokaryotic and eukaryotic host cells which may contain selective marker genes, (ab) said vector contains a DNA fragment comprising the following elements arranged in the 5'-3' direction: a eukaryotic promoter, a 5' exon of a gene, and at least the 3'-terminal exon of a gene, said arrangement of elements permitting the synthesis of a functional transcript, with a cloning site being located in an intron following the 5' exon, thereby constructing a recombinant vector; (b) transfecting eukaryotic host cells with the recombinant vector; (c) expressing the recombinant vector in the transfected host cells; (d) isolating the total RNA from the host cells in (c); (e) producing cDNA with the RNA indicated in (d), using a primer for the synthesis with reverse transcriptase, which primer is complementary to a region of an exon located downstream from the cloning site; (f) carrying out a PCR reaction with the cDNA indicated in (e) and a primer pair, the first primer being complementary to a region of the exon located upstream from the cloning site and the second primer being complementary to a region of an exon located downstream from the cloning site; (g) cloning in a vector the DNA fragment containing the additional exon(s) and obtained in the PCR reaction in (f). 2. A method for producing an exon library comprising the following steps: (a) cloning genomic DNA fragments into a cloning site of vector molecules having the following features: (aa) said vector molecules are shuttle vectors for prokaryotic and eukaryotic host cells which may contain selective marker genes, (ab) said vector molecules contain a DNA fragment comprising the following elements arranged in the 5'-3' direction: a eukaryotic promoter, a 5' exon of a gene, and at least the 3'-terminal exon of a gene, said arrangement of elements permitting the synthesis of a functional transcript, with a cloning site being located in an intron following the 5' exon, thereby constructing recombinant vector molecules; (b) transfecting eukaryotic host cells with the recombinant vector molecules; (c) expressing the DNA fragment contained in the recombinant vector in the transfected host cells; (d) isolating the total RNA from the host cells in (c); (e) producing cDNA with the total RNA indicated in (d), using a primer for the synthesis with reverse transcriptase, which primer is complementary to a region of an exon located downstream from the cloning site; (f) carrying out a PCR reaction with the cDNA indicated in (e) and a primer pair, the first primer being complementary to a region of the exon located upstream from the cloning site and the second primer being complementary to a region of an exon located downstream from the cloning site; (g) cloning in vector molecules the DNA fragments containing the additional exon(s) and obtained in the PCR reaction in (f); and (h) transforming or transfecting host cells for an exon library with the recombinant vectors obtained in (g). 3. The method as in claim 1 or 2, wherein said total RNA is poly(A) RNA. 4. The method as in claim 1 or 2 in which either primer used for the PCR reaction in (f) carries at its 5' end the recognition sequence of a desired restriction endonuclease. 5. The method as in claim 4 in which DNA fragments larger than the DNA fragment obtained only by the vector in the PCR reaction are isolated prior to cloning the DNA fragments of step (g) obtained by the PCR reaction. 6. The method as in claim 4 in which the recombinant vectors are propagated by cloning in a suitable host prior to steps (b) and/or (h). 7. The method as in claim 6, wherein said host is E. coli. 8. The method as in claim 4 in which the eukaryotic promoter is a strong promoter. 9. The method as in claim 8, wherein said promoter is LTR of RSV (Rous Sarcoma Virus). 10. The method as in claim 4 in which the exons integrated into the vector are derived from a rat insulin gene. 11. The method as in claim 4 in which the genomic DNA fragments are derived from a chromosomal region to be assayed. 12. The method as in claim 4 in which the genomic DNA fragments are derived from a genomic gene library. 13. A method for selectively cloning exons comprising the following steps: (a) cloning a genomic DNA fragment to be assayed for exons into a cloning site of a vector having the following features: (aa) said vector is a shuttle vector for prokaryotic and eukaryotic host cells which may contain selective marker genes, (ab) said vector contains a DNA fragment comprising the following elements arranged in the 5'-3' direction: an LTR promoter of RSV (Rous Sarcoma Virus), a 5' exon of a rat insulin gene, and at least the 3'-terminal exon of a rat insulin gene, said arrangement of elements permitting the synthesis of a functional transcript, with a cloning site being located in an intron following the 5' exon, thereby constructing a recombinant vector; (b) transfecting eukaryotic host cells with the recombinant vector; (c) expressing the recombinant vector in the transfected host cells; (d) isolating the poly(A) RNA from the host cells in (c); (e) producing cDNA with the RNA indicated in (d), using a primer for the synthesis with reverse transcriptase, which primer is complementary to a region of an exon located downstream from the cloning site; (f) carrying out a PCR reaction with the cDNA indicated in (e) and a primer pair, the first primer being complementary to a region of the exon located upstream from the cloning site and the second primer being complementary to a region of an exon located downstream from the cloning site; (g) cloning in a vector the DNA fragment containing the additional exon(s) and obtained in the PCR reaction in (f). 14. A method for producing an exon library comprising the following steps: (a) cloning genomic DNA fragments into a cloning site of vector molecules having the following features: (aa) said vector molecules are shuttle vectors for prokaryotic and eukaryotic host cells which may contain selective marker genes, (ab) said vector molecules contain a DNA fragment comprising the following elements arranged in the 5'-3' direction: an LTR promoter of RSV (Rous Sarcoma Virus), a 5' exon of a rat insulin gene, and at least the 3'-terminal exon of a rat insulin gene, said arrangement of elements permitting the synthesis of a functional transcript, with a cloning site being located in an intron following the 5' exon, thereby constructing recombinant vector molecules; (b) transfecting eukaryotic host cells with the recombinant vector molecules; (c) expressing the DNA fragment contained in the recombinant vector in the transfected host cells; (d) isolating the poly(A) RNA from the host cells in (c); (e) producing cDNA with the total RNA indicated in (d), using a primer for the synthesis with reverse transcriptase, which primer is complementary to a region of an exon located downstream from the cloning site; (f) carrying out a PCR reaction with the cDNA indicated in (e) and a primer pair, the first primer being complementary to a region of the exon located upstream from the cloning site and the second primer being complementary to a region of an exon located downstream from the cloning site; (g) cloning in vector molecules the DNA fragments containing the additional exon(s) and obtained in the PCR reaction in (f); and (h) transforming or transfecting host cells for an exon library with the recombinant vectors obtained in (g). 15. The method as in claim 13 or 14 in which either primer used for the PCR reaction in (f) carries at its 5' end the recognition sequence of a desired restriction endonuclease. 16. The method as in claim 13 or 14 in which DNA fragments larger than the DNA fragment obtained only by the vector in the PCR reaction are isolated prior to cloning the DNA fragments of step (g) obtained by the PCR reaction. 17. An exon library obtainable by the method as in claim 2. 18. A vector having the following features: (a) said vector is a shuttle vector for prokaryotic and eukaryotic host cells which may contain selective marker genes; (b) said vector contains a DNA fragment comprising the following elements arranged in the 5'-3' direction: a eukaryotic promoter, a 5' exon of a gene, and at least the 3'-terminal exon of a gene, said arrangement of elements permitting the synthesis of a functional transcript, with a polylinker being located in an intron following the 5' exon. 19. The vector as in claim 18 containing a genomic DNA fragment inserted into its polylinker. 20. The vector as in claim 18 or 19 in which the eukaryotic promoter is a strong promoter. 21. The vector as in claim 20, wherein said promoter is LTR of RSV (Rous Sarcoma Virus). 22. The vector as in claim 18 in which the exons integrated into the vector are derived from a rat insulin gene. 23. The vector as in claim 18 in which the genomic DNA fragment is derived from a chromosomal region to be assayed. 24. The vector as in claim 18 in which the genomic DNA fragments are derived from a genomic gene library. 25. A vector having the following features: (a) said vector is a shuttle vector for prokaryotic and eukaryotic host cells which may contain selective marker genes; (b) said vector contains a DNA fragment comprising the following elements arranged in the 5'-3' direction: an LTR promoter of RSV (Rous Sarcoma Virus) a 5' exon of a rat insulin gene, and at least the 3'-terminal exon of a rat insulin gene, a polylinker being located in an intron following the 5' exon, a genomic DNA fragment inserted into said polylinker, said arrangement of elements permitting the synthesis of a functional transcript. 26. A host organism transfected or transformed with a vector as in claim 18.

Details for Patent 5,252,475

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2010-10-12
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2010-10-12
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2010-10-12