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Gene trap mutagenesis of hnRNP A2/B1: a cryptic 3' splice site in the neomycin resistance gene allows continued expression of the disrupted cellular gene.

Roshon M, DeGregori JV, Ruley HE - BMC Genomics (2003)

Bottom Line: The mutation was selected for analysis because it occurred in a highly expressed gene and yet did not produce obvious phenotypes following germline transmission.This results in only a modest disruption of hnRNPA2/B1 gene expression.Expression of the occupied hnRNP A2/B1 gene and utilization of the viral poly(A) site are consistent with an exon definition model of pre-mRNA splicing.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Room AA4210 MCN, Vanderbilt University School of Medicine, 1161 21st Ave South, Nashville, TN, 37232-2363, USA. mroshon@carolinas.org

ABSTRACT

Background: Tagged sequence mutagenesis is a process for constructing libraries of sequenced insertion mutations in embryonic stem cells that can be transmitted into the mouse germline. To better predict the functional consequences of gene entrapment on cellular gene expression, the present study characterized the effects of a U3Neo gene trap retrovirus inserted into an intron of the hnRNP A2/B1 gene. The mutation was selected for analysis because it occurred in a highly expressed gene and yet did not produce obvious phenotypes following germline transmission.

Results: Sequences flanking the integrated gene trap vector in 1B4 cells were used to isolate a full-length cDNA whose predicted amino acid sequence is identical to the human A2 protein at all but one of 341 amino acid residues. hnRNP A2/B1 transcripts extending into the provirus utilize a cryptic 3' splice site located 28 nucleotides downstream of the neomycin phosphotransferase start codon. The inserted Neo sequence and proviral poly(A) site function as an 3' terminal exon that is utilized to produce hnRNP A2/B1-Neo fusion transcripts, or skipped to produce wild-type hnRNP A2/B1 transcripts. This results in only a modest disruption of hnRNPA2/B1 gene expression.

Conclusions: Expression of the occupied hnRNP A2/B1 gene and utilization of the viral poly(A) site are consistent with an exon definition model of pre-mRNA splicing. These results reveal a mechanism by which U3 gene trap vectors can be expressed without disrupting cellular gene expression, thus suggesting ways to improve these vectors for gene trap mutagenesis.

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HnRNP A2/B1 expression in tissues and cells from 1B4 homozygous and wild type mice. (A) Analysis of hnRNP A2/B1 expression in wild type (+/+) and homozygous mutant (-/-) mice. Approximately 20 μg of total RNA was fractionated on a 1% formaldehyde agarose gel and hybridized to a 32P labelled probe corresponding to the flanking sequence isolated by inverse PCR. The migration of the 18S ribosomal RNA is indicated. (B) Analysis of hnRNP A2/B1 expression in embryonic fibroblast cell lines derived from wild type (+/+), heterozygous (+/-), and homozygous mutant (-/-) mice. Approximately 20 μg of total RNA was fractionated on a 1% formaldehyde agarose gel and hybridized to a 32P labelled probe corresponding to hnRNP A2/B1 exons located 3' of the site of provirus integration. Levels of GAPDH transcripts were used as an internal control for RNA loading. Homozygous mutant cells displayed a two-fold reduction in hnRNPA2/B1 transcript levels, as determined by phosphoimager analysis.
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Figure 4: HnRNP A2/B1 expression in tissues and cells from 1B4 homozygous and wild type mice. (A) Analysis of hnRNP A2/B1 expression in wild type (+/+) and homozygous mutant (-/-) mice. Approximately 20 μg of total RNA was fractionated on a 1% formaldehyde agarose gel and hybridized to a 32P labelled probe corresponding to the flanking sequence isolated by inverse PCR. The migration of the 18S ribosomal RNA is indicated. (B) Analysis of hnRNP A2/B1 expression in embryonic fibroblast cell lines derived from wild type (+/+), heterozygous (+/-), and homozygous mutant (-/-) mice. Approximately 20 μg of total RNA was fractionated on a 1% formaldehyde agarose gel and hybridized to a 32P labelled probe corresponding to hnRNP A2/B1 exons located 3' of the site of provirus integration. Levels of GAPDH transcripts were used as an internal control for RNA loading. Homozygous mutant cells displayed a two-fold reduction in hnRNPA2/B1 transcript levels, as determined by phosphoimager analysis.

Mentions: To test whether the 1B4 provirus disrupts expression of the hnRNP A2/B1 gene, RNA from wild-type mice and mice homozygous for the 1B4 provirus were analyzed by Northern blot hybridization, using hnRNP A2/B1 cDNA probes derived from sequences upstream and downstream of the integration site. All tissues from wild type mice expressed a single, 1.8 kb transcript, consistent with previous studies [36,37]. Mice homozygous for the 1B4 provirus expressed the 1.8 kb transcript as well as an additional, larger transcript (Figure 4). The size of this larger transcript, approximately 2.3 kb as compared to the migration of 18S and 28S RNAs, is consistent with fusion of upstream hnRNP A2/B1 exons to the Neo gene. Sequences derived from hnRNPA2/B1 sequences downstream of the site of integration also hybridized to the 1.8 kb transcript in both wild type and 1B4 homozygous mice indicating that transcription of full-length hnRNP A2/B1 transcripts is not completely disrupted by the 1B4 provirus.


Gene trap mutagenesis of hnRNP A2/B1: a cryptic 3' splice site in the neomycin resistance gene allows continued expression of the disrupted cellular gene.

Roshon M, DeGregori JV, Ruley HE - BMC Genomics (2003)

HnRNP A2/B1 expression in tissues and cells from 1B4 homozygous and wild type mice. (A) Analysis of hnRNP A2/B1 expression in wild type (+/+) and homozygous mutant (-/-) mice. Approximately 20 μg of total RNA was fractionated on a 1% formaldehyde agarose gel and hybridized to a 32P labelled probe corresponding to the flanking sequence isolated by inverse PCR. The migration of the 18S ribosomal RNA is indicated. (B) Analysis of hnRNP A2/B1 expression in embryonic fibroblast cell lines derived from wild type (+/+), heterozygous (+/-), and homozygous mutant (-/-) mice. Approximately 20 μg of total RNA was fractionated on a 1% formaldehyde agarose gel and hybridized to a 32P labelled probe corresponding to hnRNP A2/B1 exons located 3' of the site of provirus integration. Levels of GAPDH transcripts were used as an internal control for RNA loading. Homozygous mutant cells displayed a two-fold reduction in hnRNPA2/B1 transcript levels, as determined by phosphoimager analysis.
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Related In: Results  -  Collection

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Figure 4: HnRNP A2/B1 expression in tissues and cells from 1B4 homozygous and wild type mice. (A) Analysis of hnRNP A2/B1 expression in wild type (+/+) and homozygous mutant (-/-) mice. Approximately 20 μg of total RNA was fractionated on a 1% formaldehyde agarose gel and hybridized to a 32P labelled probe corresponding to the flanking sequence isolated by inverse PCR. The migration of the 18S ribosomal RNA is indicated. (B) Analysis of hnRNP A2/B1 expression in embryonic fibroblast cell lines derived from wild type (+/+), heterozygous (+/-), and homozygous mutant (-/-) mice. Approximately 20 μg of total RNA was fractionated on a 1% formaldehyde agarose gel and hybridized to a 32P labelled probe corresponding to hnRNP A2/B1 exons located 3' of the site of provirus integration. Levels of GAPDH transcripts were used as an internal control for RNA loading. Homozygous mutant cells displayed a two-fold reduction in hnRNPA2/B1 transcript levels, as determined by phosphoimager analysis.
Mentions: To test whether the 1B4 provirus disrupts expression of the hnRNP A2/B1 gene, RNA from wild-type mice and mice homozygous for the 1B4 provirus were analyzed by Northern blot hybridization, using hnRNP A2/B1 cDNA probes derived from sequences upstream and downstream of the integration site. All tissues from wild type mice expressed a single, 1.8 kb transcript, consistent with previous studies [36,37]. Mice homozygous for the 1B4 provirus expressed the 1.8 kb transcript as well as an additional, larger transcript (Figure 4). The size of this larger transcript, approximately 2.3 kb as compared to the migration of 18S and 28S RNAs, is consistent with fusion of upstream hnRNP A2/B1 exons to the Neo gene. Sequences derived from hnRNPA2/B1 sequences downstream of the site of integration also hybridized to the 1.8 kb transcript in both wild type and 1B4 homozygous mice indicating that transcription of full-length hnRNP A2/B1 transcripts is not completely disrupted by the 1B4 provirus.

Bottom Line: The mutation was selected for analysis because it occurred in a highly expressed gene and yet did not produce obvious phenotypes following germline transmission.This results in only a modest disruption of hnRNPA2/B1 gene expression.Expression of the occupied hnRNP A2/B1 gene and utilization of the viral poly(A) site are consistent with an exon definition model of pre-mRNA splicing.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Room AA4210 MCN, Vanderbilt University School of Medicine, 1161 21st Ave South, Nashville, TN, 37232-2363, USA. mroshon@carolinas.org

ABSTRACT

Background: Tagged sequence mutagenesis is a process for constructing libraries of sequenced insertion mutations in embryonic stem cells that can be transmitted into the mouse germline. To better predict the functional consequences of gene entrapment on cellular gene expression, the present study characterized the effects of a U3Neo gene trap retrovirus inserted into an intron of the hnRNP A2/B1 gene. The mutation was selected for analysis because it occurred in a highly expressed gene and yet did not produce obvious phenotypes following germline transmission.

Results: Sequences flanking the integrated gene trap vector in 1B4 cells were used to isolate a full-length cDNA whose predicted amino acid sequence is identical to the human A2 protein at all but one of 341 amino acid residues. hnRNP A2/B1 transcripts extending into the provirus utilize a cryptic 3' splice site located 28 nucleotides downstream of the neomycin phosphotransferase start codon. The inserted Neo sequence and proviral poly(A) site function as an 3' terminal exon that is utilized to produce hnRNP A2/B1-Neo fusion transcripts, or skipped to produce wild-type hnRNP A2/B1 transcripts. This results in only a modest disruption of hnRNPA2/B1 gene expression.

Conclusions: Expression of the occupied hnRNP A2/B1 gene and utilization of the viral poly(A) site are consistent with an exon definition model of pre-mRNA splicing. These results reveal a mechanism by which U3 gene trap vectors can be expressed without disrupting cellular gene expression, thus suggesting ways to improve these vectors for gene trap mutagenesis.

Show MeSH
Related in: MedlinePlus