Retrovirus-based genomic screening
View Patent ↗The present invention relates to the expression and screening of genomic DNA sequences encoding uncharacterized genes and proteins. The present invention provides systems utilizing unique features of retroviral replication to analyze uncharacterized genes derived from genomic DNA samples. In preferred embodiments, a segment of genomic DNA is inserted between 5′ and 3′ viral long terminal repeats (LTRs) in a vector (e.g., a plasmid, cosmid, or artificial chromosome vector). The resulting vector (or library of vectors containing a plurality of independent genomic sequences) is then introduced into a retroviral packaging cell. The resulting provirus or proteins expression from the provirus are then analyzed.
1. A method for analyzing genomic DNA clones comprising:
a) providing at least one vector and at least one packaging cell, wherein said at least one vector comprises retroviral 5′ and 3′ long terminal repeats flanking a genomic DNA sequence, wherein said genomic DNA sequence is from 20 kilobases to 200 kilobases in length and includes introns;
b) introducing said at least one vector into said at least one packaging cell under conditions such that a RNA molecule corresponding to said retroviral 5′ and 3′ long terminal repeats and said genomic DNA with said introns removed sequence is transcribed and said RNA molecule is packaged into said retroviral particle, wherein said introns are removed from said RNA prior to said packaging, and wherein after removal of said introns, said RNA is 11 kilobases or less in size;
c) transducing a cell line having a genome with said retroviral particle under conditions such that said RNA molecule corresponding to said retroviral 5′ and 3′ long terminal repeats flanking a genomic DNA sequence with said introns removed is reverse transcribed and inserted into the genome of said cell line as a provirus; and
d) analyzing said genomic clone in said provirus.
2. The method of claim 1 , wherein said at least one packaging cell is a 293GP cell.
3. The method of claim 1 , wherein said retroviral particle is a pseudotyped retroviral particle.
4. The method of claim 1 , wherein said cell line is selected from the group consisting of 293 cells, CHO cells, 3T3 cells, and 208F cells.
5. The method of claim 1 , wherein said at least one vector is selected from the group consisting of plasmid, cosmid, yeast artificial chromosome, and bacterial artificial chromosome vectors.
6. The method of claim 1 , wherein said genomic DNA sequence is from 50 kilobases to 100 kilobases in length.
7. The method of claim 1 , wherein said analyzing of said provirus further comprises sequencing said provirus to provide a sequenced provirus.
8. The method of claim 7 , wherein said sequenced provirus is compared to said genomic DNA sequence to determine exon and intron boundaries.
9. The method of claim 7 , wherein multiple proviruses are sequenced and compared to identify splice variants.
10. The method of claim 1 , wherein said analyzing of said genomic clone comprises amplification of said genomic clone with PCR primers.