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A non-specific effect associated with conditional transgene expression based on Cre-loxP strategy in mice.

Qiu L, Rivera-Pérez JA, Xu Z - PLoS ONE (2011)

Bottom Line: This effect was not associated with the types or the quantity of miRNAs being expressed, nor was it associated with specific target knockdown.The presence of these inverted repeats caused a high level of cell death in the ventricular zone of the embryonic brain, where Cre was expressed.Therefore, results from this Cre-loxP approach to generate inducible transgenic alleles must be interpreted with caution and conclusions drawn in previous reports may need reexamination.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Transgenes flanked by loxP sites have been widely used to generate transgenic mice where the transgene expression can be controlled spatially and temporally by Cre recombinase. Data from this approach has led to important conclusions in cancer, neurodevelopment and neurodegeneration. Using this approach to conditionally express micro RNAs (miRNAs) in mice, we found that Cre-mediated recombination in neural progenitor cells caused microcephaly in five of our ten independent transgenic lines. This effect was not associated with the types or the quantity of miRNAs being expressed, nor was it associated with specific target knockdown. Rather, it was correlated with the presence of multiple tandem transgene copies and inverted (head-to-head or tail-to-tail) transgene repeats. The presence of these inverted repeats caused a high level of cell death in the ventricular zone of the embryonic brain, where Cre was expressed. Therefore, results from this Cre-loxP approach to generate inducible transgenic alleles must be interpreted with caution and conclusions drawn in previous reports may need reexamination.

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Detection of inverted transgene arrays in transgenic lines.(A) PCR products of tail-to-tail, head-to-head and head-to-tail junctions in seven out of ten transgenic lines (see Table 1). DNA samples from different transgenic mouse lines are shown in each lane. The positions of the primers are indicated in (B) and their sequences are shown in Table 3. (B) Sequences were successfully read from some of the PCR bands shown in (A) and these bands are marked with numbers in the parentheses. Some bands contain two sequences that can be read and these are marked by two numbers in the parentheses. The bands without sequences represent those that could not be read, most likely due to multiple sequences in the band. NC is a control in the PCR reaction using the DNA from a non-transgenic mouse. Sequences obtained from the numbered bands are aligned with the expected junctional sequences from the construct. With only one exception (the #5 band), all other junctional bands had deletions, which are marked by dotted lines between the solid lines. The solid lines represent the detected sequences. The position of the junctions is marked with negative numbers for the fragment on the left and positive numbers for the fragment on the right.
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pone-0018778-g002: Detection of inverted transgene arrays in transgenic lines.(A) PCR products of tail-to-tail, head-to-head and head-to-tail junctions in seven out of ten transgenic lines (see Table 1). DNA samples from different transgenic mouse lines are shown in each lane. The positions of the primers are indicated in (B) and their sequences are shown in Table 3. (B) Sequences were successfully read from some of the PCR bands shown in (A) and these bands are marked with numbers in the parentheses. Some bands contain two sequences that can be read and these are marked by two numbers in the parentheses. The bands without sequences represent those that could not be read, most likely due to multiple sequences in the band. NC is a control in the PCR reaction using the DNA from a non-transgenic mouse. Sequences obtained from the numbered bands are aligned with the expected junctional sequences from the construct. With only one exception (the #5 band), all other junctional bands had deletions, which are marked by dotted lines between the solid lines. The solid lines represent the detected sequences. The position of the junctions is marked with negative numbers for the fragment on the left and positive numbers for the fragment on the right.

Mentions: The GFP and RFP fluorescence intensity in the brain was examined under the fluorescent microscope and different intensities of the fluorescence were graded from the weakest “+” to the strongest “+++++”. The brain weights are expressed as ratios of the average from double transgenic mice to that of the age-matched wild type mice. The average weights with standard deviations and the statistics are shown in Table S1. The levels of mRNA and miRNA were determined by real-time RT-PCR. The mRNA levels of the double transgenic mice are presented as a percentage of the wild type mice. The miRNA levels of the double transgenic mice are presented as fold increases over the background value obtained from wild typemice. The transgene copy numbers were determined by real-time PCR of the genomic DNA from single transgenic miRNA lines. The different types of junctional fragments were detected by PCR using specific primer pairs (see Table 3). Stg: single transgenic mice positive for the G/R-miRNA transgene only.Dtg: double transgenic mice positive for both the G/R-miRNA transgene and nestin-Cre. Wt: non-transgenic mice. Br: brain. Wgt: weight ratio of Dtg-to-Wt. Stg%Wt: level in Stg as a percent of Wt. Dtg%Wt: level in Dtg as a percent of Wt. Tg: transgenic. H-T: head-to-tail.H-H: head-to-head. T-T:tail-to-tail. “+” indicates positive for the specific junctional PCR product. “−” indicates negative for the specific junctional PCR product. “+*” indicates that this PCR product sequence was successfully read (see Fig. 2).


A non-specific effect associated with conditional transgene expression based on Cre-loxP strategy in mice.

Qiu L, Rivera-Pérez JA, Xu Z - PLoS ONE (2011)

Detection of inverted transgene arrays in transgenic lines.(A) PCR products of tail-to-tail, head-to-head and head-to-tail junctions in seven out of ten transgenic lines (see Table 1). DNA samples from different transgenic mouse lines are shown in each lane. The positions of the primers are indicated in (B) and their sequences are shown in Table 3. (B) Sequences were successfully read from some of the PCR bands shown in (A) and these bands are marked with numbers in the parentheses. Some bands contain two sequences that can be read and these are marked by two numbers in the parentheses. The bands without sequences represent those that could not be read, most likely due to multiple sequences in the band. NC is a control in the PCR reaction using the DNA from a non-transgenic mouse. Sequences obtained from the numbered bands are aligned with the expected junctional sequences from the construct. With only one exception (the #5 band), all other junctional bands had deletions, which are marked by dotted lines between the solid lines. The solid lines represent the detected sequences. The position of the junctions is marked with negative numbers for the fragment on the left and positive numbers for the fragment on the right.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3091857&req=5

pone-0018778-g002: Detection of inverted transgene arrays in transgenic lines.(A) PCR products of tail-to-tail, head-to-head and head-to-tail junctions in seven out of ten transgenic lines (see Table 1). DNA samples from different transgenic mouse lines are shown in each lane. The positions of the primers are indicated in (B) and their sequences are shown in Table 3. (B) Sequences were successfully read from some of the PCR bands shown in (A) and these bands are marked with numbers in the parentheses. Some bands contain two sequences that can be read and these are marked by two numbers in the parentheses. The bands without sequences represent those that could not be read, most likely due to multiple sequences in the band. NC is a control in the PCR reaction using the DNA from a non-transgenic mouse. Sequences obtained from the numbered bands are aligned with the expected junctional sequences from the construct. With only one exception (the #5 band), all other junctional bands had deletions, which are marked by dotted lines between the solid lines. The solid lines represent the detected sequences. The position of the junctions is marked with negative numbers for the fragment on the left and positive numbers for the fragment on the right.
Mentions: The GFP and RFP fluorescence intensity in the brain was examined under the fluorescent microscope and different intensities of the fluorescence were graded from the weakest “+” to the strongest “+++++”. The brain weights are expressed as ratios of the average from double transgenic mice to that of the age-matched wild type mice. The average weights with standard deviations and the statistics are shown in Table S1. The levels of mRNA and miRNA were determined by real-time RT-PCR. The mRNA levels of the double transgenic mice are presented as a percentage of the wild type mice. The miRNA levels of the double transgenic mice are presented as fold increases over the background value obtained from wild typemice. The transgene copy numbers were determined by real-time PCR of the genomic DNA from single transgenic miRNA lines. The different types of junctional fragments were detected by PCR using specific primer pairs (see Table 3). Stg: single transgenic mice positive for the G/R-miRNA transgene only.Dtg: double transgenic mice positive for both the G/R-miRNA transgene and nestin-Cre. Wt: non-transgenic mice. Br: brain. Wgt: weight ratio of Dtg-to-Wt. Stg%Wt: level in Stg as a percent of Wt. Dtg%Wt: level in Dtg as a percent of Wt. Tg: transgenic. H-T: head-to-tail.H-H: head-to-head. T-T:tail-to-tail. “+” indicates positive for the specific junctional PCR product. “−” indicates negative for the specific junctional PCR product. “+*” indicates that this PCR product sequence was successfully read (see Fig. 2).

Bottom Line: This effect was not associated with the types or the quantity of miRNAs being expressed, nor was it associated with specific target knockdown.The presence of these inverted repeats caused a high level of cell death in the ventricular zone of the embryonic brain, where Cre was expressed.Therefore, results from this Cre-loxP approach to generate inducible transgenic alleles must be interpreted with caution and conclusions drawn in previous reports may need reexamination.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Transgenes flanked by loxP sites have been widely used to generate transgenic mice where the transgene expression can be controlled spatially and temporally by Cre recombinase. Data from this approach has led to important conclusions in cancer, neurodevelopment and neurodegeneration. Using this approach to conditionally express micro RNAs (miRNAs) in mice, we found that Cre-mediated recombination in neural progenitor cells caused microcephaly in five of our ten independent transgenic lines. This effect was not associated with the types or the quantity of miRNAs being expressed, nor was it associated with specific target knockdown. Rather, it was correlated with the presence of multiple tandem transgene copies and inverted (head-to-head or tail-to-tail) transgene repeats. The presence of these inverted repeats caused a high level of cell death in the ventricular zone of the embryonic brain, where Cre was expressed. Therefore, results from this Cre-loxP approach to generate inducible transgenic alleles must be interpreted with caution and conclusions drawn in previous reports may need reexamination.

Show MeSH
Related in: MedlinePlus