scramble shrna lentiviral control vector plv[shrna]-egfp/puro-u6  (VectorBuilder GmbH)

Journal: Stem Cell Reports

Article Title: Highly Efficient Genome Modification of Cultured Primordial Germ Cells with Lentiviral Vectors to Generate Transgenic Songbirds

doi: 10.1016/j.stemcr.2021.02.015

Figure Lengend Snippet: Cultured zfPGCs Were Efficiently Transduced with a Lentiviral Vector for eGFP and Colonized Host Embryo Gonads (A) The lentiviral vector rrl-hPGK-eGFP that was used to transduce zfPGCs in vitro and generate founder birds. (B–F) Microphotographs of cultured zfPGCs that were transduced with rrl-hPGK-eGFP for 48 h. Phase-contrast (B) and eGFP expression (C) of zfPGCs are shown at low magnification. Note that almost all zfPGCs were expressing the reporter gene. In (D–F), fluorescence images of a PGC cluster show the same cells expressing eGFP (D), being immunopositive for the PGC marker EMA-1 (E), and after DAPI staining (F). The inset in (E) shows a merged eGFP- and anti-EMA-1 image of a subset of cells (indicated by the arrow). Note that eGFP-expressing cells were EMA-1 immunopositive. (G and H) In microphotographs of cryosections produced from an adult founder ovary that showed eGFP expression and blue nuclear counterstain with DAPI are presented at low (G) and high (H) magnification. Note that in the ovary eGFP-positive germ cells of different sizes display various stages of differentiation. Scale bars represent 100 μm (C, G, and H) and 20 μm (D–F). (I–K) Photographs of agarose gels after electrophoretic separation and ethidium bromide staining of molecular weight markers (left lanes) and PCR products. PCR products that were obtained using the lentiviral vector plasmid (lane 1 in I and J) are shown as positive controls. Genomic DNA (gDNA) was isolated from a founder ovary (I, lane 2) and testis (I, lane 3), and from blood samples of three F1 birds, two males (J, lanes 2 and 3) and one female (J, lane 4). The right arrowhead in (I and J) points to the hPGK-promoter-eGFP amplicon (602 bp). To verify expression of the transgene by RT-PCR (K), RNA was isolated from the brain of a wild-type bird (K, lane 1) as well as brain (K, lanes 2 and 3) and liver (K, lanes 4 and 5) samples of different F1 birds. Although transgene expression could not be detected in skeletal muscle of transgenic birds by RT-PCR, the transgene was detectable in skeletal muscle gDNA of transgenic birds (K, lanes 7 and 8), in contrast to the wild type (K, lane 6). The right arrowhead in (K) points to the eGFP-WPRE amplicon (894 bp).

Article Snippet: The lentiviral vector for eGFP under control of the human EF1α promoter was purchased from SignaGen Laboratories (Gaithersburg, MD, USA).

Techniques: Cell Culture, Transduction, Plasmid Preparation, In Vitro, Expressing, Fluorescence, Marker, Staining, Produced, Molecular Weight, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay