Human Platelet Lysate Media Supplement Supports Lentiviral Transduction and Expansion of Human T Lymphocytes While Maintaining Memory Phenotype.

Immune cell therapy has emerged as a promising approach to treat malignancies that were up until recently only treated on a palliative basis. Chimeric antigen receptor- (CAR-) modified T lymphocytes (T cells) in particular have proven to be very effective for certain hematological malignancies. The production of CAR T cells usually involves viral transduction and culture of T cells.

Mesenchymal to Epithelial Transition Mediated by CDH1 Promotes Spontaneous Reprogramming of Male Germline Stem Cells to Pluripotency.

Cultured spermatogonial stem cells (GSCs) can spontaneously form pluripotent cells in certain culture conditions. However, GSC reprogramming is a rare event that is largely unexplained. We show GSCs have high expression of mesenchymal to epithelial transition (MET) suppressors resulting in a developmental barrier inhibiting GSC reprogramming.

Ectopic POU5F1 in the male germ lineage disrupts differentiation and spermatogenesis in mice.

Expression levels of the pluripotency determinant, POU5F1, are tightly regulated to ensure appropriate differentiation during early embryogenesis. POU5F1 is also present in the spermatogonial stem cell/progenitor cell population in mice and it is downregulated as spermatogenesis progresses. To test if POU5F1 downregulation is required for SSCs to differentiate, we produced transgenic mice that ubiquitously express POU5F1 in Cre-expressing lineages.

Genome editing of the germline: broadening the discussion.

Genome editing that results in humans with precisely modified germ cells may never become practical. Nonetheless, the implications are great enough that we strongly support the idea of starting the conversation now, providing time for a broad consensus to be developed. We are confident that if diverse voices are heard, a consensus can be reached on a strategy in which societal mores are respected, the desires of parents are integrated, and the health of future generations is maximized.

Genome editing in mouse spermatogonial stem/progenitor cells using engineered nucleases.

Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease.

Transgenic modification of spermatogonial stem cells using lentiviral vectors.

The continuous production of spermatazoa throughout the reproductive lifetime of a male depends on the maintenance of a pool of progenitor cells called spermatogonial stem cells (SSCs). SSCs represent a very small fraction of the cellular population in the testes and lack definitive molecular markers for their identification. The discovery of conditions that allow one to propagate mouse SSCs in vitro essentially indefinitely has truly facilitated studies of the molecular mechanisms regulating SSC function.

Generation of a germ cell-specific mouse transgenic CHERRY reporter, Sohlh1-mCherryFlag.

Visualization of differentiating germ cells is critical to understanding the formation of primordial follicles in the ovary, and the commitment of spermatogonial stem cells to differentiation. We engineered and generated a BAC transgenic mouse line, Sohlh1-mCherryFlag (S1CF), under the direction of the native Sohlh1 promoter. Sohlh1 is a germ cell-specific gene that encodes the basic helix-loop-helix (bHLH) transcriptional regulator that is essential in oogenesis and spermatogenesis.

Development of quantitative microscopy-based assays for evaluating dynamics of living cultures of mouse spermatogonial stem/progenitor cells.

Spermatogonial stem cell (SSC) self-renewal and differentiation are required for continuous production of spermatozoa and long-term fertility. Studying SSCs in vivo remains challenging because SSCs are rare cells and definitive molecular markers for their identification are lacking. The development of a method for propagating SSCs in vitro greatly facilitated analysis of SSCs.

Dynamic expression pattern and subcellular localization of the Rhox10 homeobox transcription factor during early germ cell development.

Homeobox genes encode transcription factors that regulate diverse developmental events. The largest known homeobox gene cluster - the X-linked mouse reproductive homeobox (Rhox) cluster - harbors genes whose expression patterns and functions are largely unknown. Here, we report that a member of this cluster, Rhox10, is expressed in male germ cells.

Imatinib has deleterious effects on differentiating spermatogonia while sparing spermatogonial stem cell self renewal.

Imatinib mesylate is among a growing number of effective cancer drugs that provide molecularly targeted therapy; however, imatinib causes reproductive defects in rodents. The availability of an in vitro system for screening the effect of drugs on spermatogenesis would be beneficial. The imatinib targets, KIT and platelet derived growth factor receptor beta (PDGFRB), were shown here to be expressed in "germline stem" (GS) cell cultures that contain spermatogonia, including spermatogonial stem cells (SSCs).

Spermatogonial stem cell self-renewal requires OCT4, a factor downregulated during retinoic acid-induced differentiation.

The long-term production of billions of spermatozoa relies on the regulated proliferation and differentiation of spermatogonial stem cells (SSCs). To date only a few factors are known to function in SSCs to provide this regulation. Octamer-4 (OCT4) plays a critical role in pluripotency and cell survival of embryonic stem cells and primordial germ cells; however, it is not known whether it plays a similar function in SSCs.

Production of knockdown rats by lentiviral transduction of embryos with short hairpin RNA transgenes.

The primary method for determining the function of a gene in rodents has been to make a knockout mouse through homologous recombination in embryonic stem cells. However, with the advent of RNA interference (RNAi) technology, new methods for studying gene function are now possible in a wide array of animals. We describe a protocol for knocking down a gene of interest in vivo in rats by stably expressing a short hairpin RNA (shRNA).

New technology for an old favorite: lentiviral transgenesis and RNAi in rats.

The ability to produce targeted deletions in the mouse genome via homologous recombination has been a hallmark of mouse genetics, and has lead to the production of thousands of gene knockouts. New technologies are making it possible to disrupt gene function in many other species. This article reviews some of these methods, highlighting the powerful combination of lentiviral vectors with RNA interference (RNAi), which allows one to produce transgenic animals expressing short hairpin RNA (shRNA) to "knock down" specific gene expression.

Heritable and stable gene knockdown in rats.

The rat has served as an excellent model for studies on animal physiology and as a model for human diseases such as diabetes and alcoholism; however, genetic studies have been limited because of the inability to knock out genes. Our goal was to produce heritable deficiencies in specific gene function in the rat using RNA interference to knock down gene expression in vivo. Lentiviral-mediated transgenesis was used to produce rats expressing a short hairpin RNA targeting Dazl, a gene expressed in germ cells and required for fertility in mice.