Robust generation of transgenic mice by simple hypotonic solution mediated delivery of transgene in testicular germ cells

Abstract

Our ability to decipher gene sequences has increased enormously with the advent of modern sequencing tools, but the ability to divulge functions of new genes have not increased correspondingly. This has caused a remarkable delay in functional interpretation of several newly found genes in tissue and age specific manner, limiting the pace of biological research. This is mainly due to lack of advancements in methodological tools for transgenesis. Predominantly practiced method of transgenesis by pronuclear DNA-microinjection is time consuming, tedious, and requires highly skilled persons for embryo-manipulation. Testicular electroporation mediated transgenesis requires use of electric current to testis. To this end, we have now developed an innovative technique for making transgenic mice by giving hypotonic shock to male germ cells for the gene delivery. Desired transgene was suspended in hypotonic Tris-HCl solution (pH 7.0) and simply injected in testis. This resulted in internalization of the transgene in dividing germ-cells residing at basal compartment of tubules leading to its integration in native genome of mice. Such males generated transgenic progeny by natural mating. Several transgenic animals can be generated with minimum skill within short span of time by this easily adaptable novel technique.

Multiplex shRNA Screening of Germ Cell Development by in Vivo Transfection of Mouse Testis

“…recently developed a novel method for direct transfection of testicular germ cells that provides a nonviral, nonsurgical alternative for nucleic acid delivery. This technique uses a buffered salt solution to generate an osmotic gradient that drives water and dissolved linear DNA into the germ cells of the testis at a reasonably high rate (Usmani et al 2016). Once inside the cells, the DNA integrates spontaneously and randomly into the genome at a concentration-dependent rate, presumably through nonhomologous end joining during routine DNA repair.…”

Possible Production of Genome-Edited Animals Using Gene-Engineered Sperm

“…On the contrary, He et al [53] demonstrated that the transgene transmission rate from F0 to F1 generation was 37%, suggesting that the transgene transmission rate was similar to the Mendelian law of inheritance. In 2007 and onward, attempts to improve IIGT systems were made by several laboratories to enhance the gene delivery efficiency [54][55][56][57][58][59][60][61][62][63][64][65][66][67]. In the following sections, we will describe several examples [(v) to (x)] about the improvement of IIGT, in vitro assessment for gene expression after IIGT or possible mechanism underlying IIGT.…”

“…To overcome this difficulty, they developed an alternative technique for making Tg mice by hypotonic shocking male germ cells for gene delivery. According to Usmani et al [65], treatment with hypotonic Tris-HCl solution reduced osmolarity and led to hypotonic-swelling of germ cells. The hypotonic-swelling eventually killed the cells with increased hypotonicity, but led to the uptake of surrounding molecules such as nucleosides inside the cell.…”

“…The authors hypothesized that a hypotonic Tris-HCl solution at a certain hypotonic concentration might allow the germ cells to internalize the surrounding solutes like DNA in vivo without being killed and the sperm produced from transfected germ cells may carry a desired DNA fragment (transgene) to generate Tg animals. Usmani et al [65] suspended the linearized plasmid DNA (transgenes) in hypotonic Tris-HCl solution (pH 7.0) and simply performed IIGT to internalize the injected transgenes in the genome of spermatogonia residing at basal compartment of tubules. As a result, such males successfully generated Tg progeny by natural mating.…”

“…This technique is easy and simple and does not require expensive apparatuses like electroporators. Usmani et al [65] proposed that such a procedure enables researchers to generate their own Tg animals, instead of outsourcing, and would drastically minimize the time required for studies on functional genomics.…”