Non-Integrative Lentivirus Drives High-Frequency cre-Mediated Cassette Exchange in Human Cells

Recombinase mediated cassette exchange (RMCE) is a two-step process leading to genetic modification in a specific genomic target sequence. The process involves insertion of a docking genetic cassette in the genome followed by DNA transfer of a second cassette flanked by compatible recombination signals and expression of the recombinase. Major technical drawbacks are cell viability upon transfection, toxicity of the enzyme, and the ability to target efficiently cell types of different origins. To overcome such drawbacks, we developed an RMCE assay that uses an integrase-deficient lentivirus (IDLV) vector in the second step combined with promoterless trapping of double selectable markers. Additionally, recombinase expression is self-limiting as a result of the exchangeable reaction, thus avoiding toxicity. Our approach provides proof-of-principle of a simple and novel strategy with expected wide applicability modelled on a human cell line with randomly integrated copies of a genetic landing pad. This strategy does not present foreseeable limitations for application to other cell systems modified by homologous recombination. Safety, efficiency, and simplicity are the major advantages of our system, which can be applied in low-to-medium throughput strategies for screening of cDNAs, non-coding RNAs during functional genomic studies, and drug screening

DNA methylation-based classification of sinonasal tumors

The diagnosis of sinonasal tumors is challenging due to a heterogeneous spectrum of various differential diagnoses as well as poorly defined, disputed entities such as sinonasal undifferentiated carcinomas (SNUCs). In this study, we apply a machine learning algorithm based on DNA methylation patterns to classify sinonasal tumors with clinical-grade reliability. We further show that sinonasal tumors with SNUC morphology are not as undifferentiated as their current terminology suggests but rather reassigned to four distinct molecular classes defined by epigenetic, mutational and proteomic profiles. This includes two classes with neuroendocrine differentiation, characterized by IDH2 or SMARCA4/ARID1A mutations with an overall favorable clinical course, one class composed of highly aggressive SMARCB1-deficient carcinomas and another class with tumors that represent potentially previously misclassified adenoid cystic carcinomas. Our findings can aid in improving the diagnostic classification of sinonasal tumors and could help to change the current perception of SNUCs

DNA methylation-based classification of sinonasal tumors

The diagnosis of sinonasal tumors is challenging due to a heterogeneous spectrum of various differential diagnoses as well as poorly defined, disputed entities such as sinonasal undifferentiated carcinomas (SNUCs). In this study, we apply a machine learning algorithm based on DNA methylation patterns to classify sinonasal tumors with clinical-grade reliability. We further show that sinonasal tumors with SNUC morphology are not as undifferentiated as their current terminology suggests but rather reassigned to four distinct molecular classes defined by epigenetic, mutational and proteomic profiles. This includes two classes with neuroendocrine differentiation, characterized by IDH2 or SMARCA4/ARID1A mutations with an overall favorable clinical course, one class composed of highly aggressive SMARCB1-deficient carcinomas and another class with tumors that represent potentially previously misclassified adenoid cystic carcinomas. Our findings can aid in improving the diagnostic classification of sinonasal tumors and could help to change the current perception of SNUCs

DNA methylation-based classification of sinonasal tumors

The diagnosis of sinonasal tumors is challenging due to a heterogeneous spectrum of various differential diagnoses as well as poorly defined, disputed entities such as sinonasal undifferentiated carcinomas (SNUCs). In this study, we apply a machine learning algorithm based on DNA methylation patterns to classify sinonasal tumors with clinical-grade reliability. We further show that sinonasal tumors with SNUC morphology are not as undifferentiated as their current terminology suggests but rather reassigned to four distinct molecular classes defined by epigenetic, mutational and proteomic profiles. This includes two classes with neuroendocrine differentiation, characterized by IDH2 or SMARCA4/ARID1A mutations with an overall favorable clinical course, one class composed of highly aggressive SMARCB1-deficient carcinomas and another class with tumors that represent potentially previously misclassified adenoid cystic carcinomas. Our findings can aid in improving the diagnostic classification of sinonasal tumors and could help to change the current perception of SNUCs

Nurses' perceptions of aids and obstacles to the provision of optimal end of life care in ICU

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PCR analysis of genomic DNA isolated to verify RMCE reaction.

<p>PCR analysis of genomic DNA extracted from the HEK293A cells (lane 1 and 7), or transfected with pKAS (293AKAS) (lanes 2 to 5 and 8 to 11). Cells were transduced with control virus IDLV.CMVeGFP (lanes 2 and 8), IDLV.TRINA (lanes 4, 5, 10 and 11), or non-transduced (lanes 3 and 9). Positive control is DNA from pKAS (lanes 6 and 12). The 1250 bp band is indicative of RMCE between KAS and TRINA cassettes and the 1450 bp corresponds to integrated pKAS band. IDLV.TRINA/10 and IDLV.TRINA/20 correspond to DNA obtained after from cultures selected during10 and 20 days with hygromycin respectively. The sizes of the DNA fragments in the marker line M are indicated.</p

Randomly chromosomal integrated copies of the genetic landing pad KAS are targeted by IDLV expressing the TRINA cassette.

<p>HEK293A cells resistant to G418 (293AKAS) were pooled and transduced with low MOI (2 transduction unit/cell) of IDLV-TRINA. (A) Transduced cells were selected with hygromycin and the colonies counted. The figure shows stained plates after selection of cells MOCK-transduced (LV.eGFP, left) or transduced with IDLV-TRINA (center) or IDLV-Cre (right). (B) Quantification of RMCE frequency in 293AKAS cells by culturing under selection conditions with hygromycin upon transduction at different MOI with IDLV-TRINA . (C) Quantification of RMCE frequency in 293AKAS cells by FACS analysis of ChFP+ upon transduction at different MOI with IDLV-TRINA as in B. Triplicate cultures were transduced, FACS analyzed in triplicate. Average +/−SD is represented. (D) Western blot to detect cre expression in pooled 293AKAS cells at different points after transduction with IDLV.TRINA (MOI 2 TU/cell). The alpha-tubulin protein was used as a loading control (bottom panel). Results in A and C are from one representative experiment out of three. ETF: Effective Targeting Frecquency: ratio between hygro^R (B) or ChFP+ (C) and the number of total G418^R cells plated for transduction.</p

Plasmid-mediated RMCE by transient transfection.

<p>(A) Hygromycin-resistant colonies obtained upon transfection of HEK293A cells with the indicated plasmids containing the genetic landing pad (pKAS) or the transfer plasmid (pTRINA) expressing cre-recombinase at two different relative amounts (low and high [pTRINA]). Colonies were stained after ten days under conditions of selection. (B) Microphotographs under brightfields (left) and fluorescent light (right) of the transfected cultures with the indicated plasmids. (C) Number of cells expressing cherry fluorescent protein (ChFP+) upon RMCE on HEK293A cells measured by FACS analysis. Variable amounts of pKAS were used with a constant amount of pTRINA as is indicated. Transfections were made in duplicate, and the results shown in both A and B are from one representative experiment out of three.</p

Cre recombinase-mediated insertion and cassette exchange strategy.

<p>Structure of the genetic landing pad (KAS) and the incoming construct (LV.TRINA) showing the more relevant elements: promoters PGK, EF-1α and SV40 in the neo cassette (black arrows), reporter genes (coloured boxes), recombinase cre (green box), translation starting point (bended arrows), and polyA signals (crossed boxes). Triangles represent the loxP (black) and lox2272 (white) sites. ROSA26 homology arms are pink-colored and lentiviral LTRs drawn as clear boxes flanking the KAS and TRINA cassette respectively. For simplicity the CHYSEL and multicloning site downstream of the neo cassette are not indicated. The expected resulting endpoints after RMCE reaction are depicted below. Symbols are kept for simplicity. ChFP, cherry fluorescent protein; LTR, long terminal repeat.</p