fluorescent markers chromosome tracking cell cycle tracking (meiosis, etc.) spindle formation tracking fluorescently-labeled chromosomes parthenogenesis activation microinjection automated microinjection automated microfluidic microinjection intracytoplasmic sperm injection ooplasm transfer ooplasm xenotransfer direct DNA injection direct chromsome (cDNA) injection artificial chromosome injection germ cell xenotransplantation testicular tissue xenotransplantation sperm production testis-mediated gene therapy germ cell gene therapy seminiferous tubule genetic transformation electroporation lipofection uniquely fluorescently-labeled chromosomes + micromanipulator separation of chromosomes physical intervention during meiosis Non-invasive deactivation and spatial control of intracellular chromosome with magnetic nanoparticle http://s-space.snu.ac.kr/bitstream/10371/118542/1/000000136035.pdf In this study, we demonstrate a novel strategy to non-invasively regulate chromosome activity by targeting genetic regulation materials (i.e., oocyte-specific linker histone H1 protein) in live oocyte with the functionalized magnetic nanoparticles and to spatially control the targeted chromosomes by a remote magnetic field. To this end, bacterial magnetic nanoparticles (BMPs, ~50 nm) produced by magnetotactic bacteria (Magnetosome) were used because of their peculiar features, such as high magnetism, high dispersal ability in an aqueous media and good biocompatibility. In addition, BMPs can effectively be conjugated to the diverse biomolecules due to the abundance of amine groups on particles surrounded by lipid membrane. Recently, although human stem cell was generated by somatic cell nuclear transfer (SCNT), the enucleation process is still important factor in SCNT. It is well known that the invasive enucleation (removal of human oocyte genome) leads to the failure of embryonic development after genome exchange due to the loss of meiosis-specific factors associated with the spindle removal during physical enucleation process. Thus, we suggest the non-invasive method for SCNT without enucleation, by targeting chromosome with H1-BMPs. magnetic labeling of chromosomes magnetic nanoparticles functionalization such as with antibodies (can help with magnetic labeling of chromosomes) fluorescent markers magnetic control spermatid injection spermatocyte injection ?? adult gamete cell (stem cell?) injection ?? cell fusion electrofusion somatic cell nuclear transfer nuclear transfer pronuclear injection cell cloning post-fertilization genetic modification - low efficiency will mean many discarded attempts blastomere zygote ooctyes time since harvested storage protocol liquid media composition temperature "Temperature-induced orientation instability during meiosis: an experimental analysis" fluorescent markers fluorescent markers of fertility sperm cells time since harvested storage protocol liquid media composition temperature fluorescent markers of genetic modification fluorescent markers of sperm cell fertilization potential cell fusion of somatic cells to oocytes use overexpression of STRA8, BOULE, and DAZL in the somatic cell interspecies somatic cell nuclear transfer (iSCNT) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3787369/ "iSCNT for cloning animals works only for species that can interbreed" human-bovine Blastocyst formation, karyotype, and mitochondrial DNA of interspecies embryos derived from nuclear transfer of human cord fibroblasts into enucleated bovine oocytes. Evaluation of the embryonic preimplantation potential of human adult somatic cells via an embryo interspecies bioassay using bovine oocytes. Activation of human embryonic gene expression in cytoplasmic hybrid embryos constructed between bovine oocytes and human fibroblasts. human-ovine Enucleated ovine oocyte supports human somatic cells reprogramming back to the embryonic stage. upregulation of Oct4, Sox2, and Nanog human-goat Fates of donor and recipient mitochondrial DNA during generation of interspecies SCNT-derived human ES-like cells. Ooplast transfer of triploid pronucleus zygote improve reconstructed human-goat embryonic development https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238477/ Simple, fast, and efficient method of manual oocyte enucleation using a pulled Pasteur pipette Study of a microfluidic chip integrating single cell trap and 3D stable rotation manipulation Chemically assisted somatic cell nuclear transfer without micromanipulator in the goat: effects of demecolcine, cytochalasin-B, and MG-132 The effect of amniotic membrane stem cells as donor nucleus on gene expression in reconstructed bovine oocytes Towards on-chip single cell manipulation of trap and rotation in vitro sexual genetic recombination is this really necessary? just mix the chromosomes and pick one from each parent later use DNA synthesis for actual chromosomal mixing recombination chromosomal crossover recombination mammalian meiotic recombination useful things: - in vitro protocol for mammalina meiotic recombination (or equivalent) - xenotransplantation of chromosomes into a cell body capable of appropriate meiotic recombination cell fusion? direct injection of cDNA ? encapsulated cDNA in liposomes etc.? The choice in meiosis – defining the factors that influence crossover or non-crossover formation http://jcs.biologists.org/content/124/4/501 ATR is required to complete meiotic recombination in mice https://www.biorxiv.org/content/biorxiv/early/2017/05/03/133744.full.pdf Understanding and manipulating meiotic recombination in plants http://www.plantphysiol.org/content/173/3/1530 chromosome crossover formation Advances towards controlling meiotic recombination for plant breeding https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712510/ "Arabidopsis genes involved in meiotic crossover frequency" FANCM helicase RTR complex FIGL1 helicase Class I CO pathway non-CG methylation H3K9me2 Anti-crossover (MSH2) "mutation of the MSH2 gene attenuates heterozygosity-mediated crossover suppression and results in a 40% increase in crossover rate (Emmanuel et al., 2006)" mutation of anti-crossover genes (9x crossovers) extra HEI10 (2x crossovers) MSH2 mutation (crossover in heterozygous state) Loss of non-CG me (crossovers in heterochromatin) "CRISPR based tools can be further modified with either meiotic specific expression, or fusion with a meiotic protein as an effector, to induce meiotic recombination at a specific target site and potentially increase genetic and epigenetic variations of favorable traits that involve linked alleles such as clustered plant immunity genes (Deng et al., 2017)." oocytes are arrested at the dictyate stage of meiosis Spo11 to generate meiotic DSBs (double strand breaks) MEI14 and REC114 Mei1 found during "Mouse mutants from chemically mutagenized embryonic stem cells" yeast meiotic recombination of human chromosomes, then do nuclear transfer into human cells? Specific genetic modifications of domestic animals by gene targeting and animal cloning https://rbej.biomedcentral.com/articles/10.1186/1477-7827-1-103 Dissecting the mammalian synaptonemal complex using targeted mutations https://link.springer.com/article/10.1007/s10577-007-1142-1 Sequence variants in the RNF212 gene associate with genome-wide recombination rate A novel mouse synaptonemal complex protein is essential for loading of central element proteins, recombination, and fertility Cohesin SMC1β protects telomeres in meiocytes Interplay between synaptonemal complex, homologous recombination, and centromeres during mammalian meiosis oocyte shrinking (within its outer coating) by placing it in a sugar solution mouse sperm generation cell culture Sato, T. et al. In vitro production of functional sperm in cultured neonatal mouse testes. Nature 471, 504–7 (2011). mouse oocyte generation cell culture Complete in vitro generation of fertile oocytes from mouse primordial germ cells oocyte activation factors zygote transfection (after fertilization and recombination) (30 hour window in humans before blastomeres are formed) https://www.nature.com/articles/srep02847 injected 208 zygotes, GFP detected in 36% of embryos Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA https://www.nature.com/articles/srep08256 cytoplasmic hybrid meiosis cell cycle arrest (use nocodazole treatment) selective enucleation Developmental potential of selectively enucleated immature mouse oocytes upon nuclear transfer Sucrose pretreatment for enucleation: An efficient and non‐damage method for removing the spindle of the mouse MII oocyte In vitro parthenogenetic development of mouse oocytes following reciprocal transfer of the chromosome spindle between in vivo-matured oocytes and in vitro-matured oocytes These results indicate that the low developmental competence of in vitro-matured oocytes from mouse preantral follicles after activation is caused by the cytoplasmic component rather than the nuclear component. The use of micromanipulation methods as a tool to prevention of transmission of mutated mitochondrial DNA Optimal timing for oocyte denudation and intracytoplasmic sperm injection Germ cell transplantation from large domestic animals into mouse testes later stages of donor‐derived spermatogenesis were not observed Spermatogenesis following male germ-cell transplantation stem cells isolated from testes of donor male mice will repopulate sterile testes when injected into seminiferous tubules Orthotopic testicular transplantation in mice http://www.reproduction-online.org/content/139/2/447.short revascularized orthotopic testicular transplantation transplanted testes showed active spermatogenesis and normal structure of epididymis at 4 and 5 weeks Homologous testis transplantation in dogs prednisolone + cyclosporin A Transplantation of the testis; from the past to the present (1996) Homologous testis transplantation has never become a subject of great interest, probably for ethical reasons. This possible treatment for hypogonadism, however, has been developed experimentally and has been performed in man only in Russia and China, evidently with success. Primate spermatogonial stem cells colonize mouse testes https://academic.oup.com/biolreprod/article/64/5/1409/2723365 differentiation of germ cells toward the lumen of the tubule and production of spermatozoa did not occur Identifying genes important for spermatogonial stem cell self-renewal and survival Long-term survival of human spermatogonial stem cells in mouse testes Propagation of human spermatogonial stem cells in vitro Accelerated maturation of primate testis by xenografting into mice Birth of offspring following transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination Slow freezing, but not vitrification supports complete spermatogenesis in cryopreserved, neonatal sheep testicular xenografts Xenografting of testicular tissue pieces: 12 years of an in vivo spermatogenesis system Fragments of monkey and goat testes were xenografted into humans and surgeons claimed that it restored physical and intellectual abilities (Setchell 1990). However, Voronoff’s statements were refuted and rejection of donor testicular tissue was demonstrated (Gunn & Seddon 1930, Setchell 1990). Subcutaneous xenografting of rat, pig, sheep and peccary testicular cell suspensions formed functional testicular tissue. This procedure could be used to study testicular morphogenesis and cell interactions, but the onset of spermatogenesis is delayed when compared with xenotransplantation of testicular fragment (Gassei et al. 2006, Honaramooz et al. 2007, Arregui et al. 2008a, Campos-Junior et al. 2013) Sato et al. 2011 In vitro production of functional sperm in cultured neonatal mouse testes. Nature 471 504–507. (doi:10.1038/nature09850) Stukenborg et al. 2009 New horizons for in vitro spermatogenesis? An update on novel three-dimensional culture systems as tools for meiotic and post-meiotic differentiation of testicular germ cells Abu Elhija et al. 2012 Differentiation of murine male germ cells to spermatozoa in a soft agar culture system Mouse round spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo-developed mature oocytes Can we induce spermatogenesis in the domestic cat using an in vitro tissue culture approach? (2018) Spermatogonial stem cell autotransplantation and germline genomic editing: a future cure for spermatogenic failure and prevention of transmission of genomic diseases https://academic.oup.com/humupd/article/22/5/561/1749854 genetic editing in spermatgonial stem cells transplantation of the spermatogonial stem cells back to the male testis/seminiferous tubules followed by natural generation of modified sperm Wu Y, Zhou H, Fan X, Zhang Y, Zhang M, Wang Y, Xie Z, Bai M, Yin Q, Liang D et al. Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells. Cell Res 2015;25:67–79. Chapman KM, Medrano GA, Jaichander P, Chaudhary J, Waits AE, Nobrega MA, Hotaling JM, Ober C, Hamra FK. Targeted germline modifications in rats using CRISPR/Cas9 and spermatogonial stem cells. Cell Rep 2015;10:1828–1835. Sato T, Sakuma T, Yokonishi T, Katagiri K, Kamimura S, Ogonuki N, Ogura A, Yamamoto T, Ogawa T. Genome editing in mouse spermatogonial stem cell lines using TALEN and double-nicking CRISPR/Cas9. Stem Cell Reports 2015;5:75–82. Interestingly, a recent article reports successful genome editing of mouse SSCs with the CRISPR-Cas9 system (Wu et al., 2015). Transplantation of the genetically modified SSCs led to fertile offspring, in which a Crygc mutation causing cataracts was corrected. To our knowledge, this is the first report that describes CRISPR-Cas9-mediated genome editing in SSCs in combination with SSCT, thereby preventing diseases in the offspring. Furthermore, as the transplanted SSCs were the cell lines derived from isolated and corrected single cells, this method can generate healthy descendants at 100% efficiency, thereby averting the problem of mosaicism. In addition to this pioneering work, two other recent articles also provide the proof of concept by showing CRISPR-Cas9 and SSCT-induced germline transmission in rodents (Chapman et al., 2015; Sato et al., 2015). Hence, SSCT and the CRISPR-Cas9 system can be well combined in the future to prevent the transmission of inheritable diseases to the offspring. Human SSCs may change (epi)genetically when exposed to an in vitro environment. Yet, there is evidence of genetic stability of cultured human SSCs (Nickkholgh et al., 2014). DNA methylation of maternal and paternal imprinted genes in uncultured murine SSCs did not alter after transplantation (Goossens et al., 2009; Wu et al., 2012), whereas cultured human SSCs showed changes in DNA methylation in some selected regions of maternal and paternal imprinted genes (Nickkholgh et al., 2014). In addition, a major clinical drawback of germline therapy in fertile carriers of diseases remains that the patients have to undergo local irradiation to deplete the testis of endogenous mutated spermatogonia before SSCT. Otherwise, the testis of the recipient father would produce two populations of sperm cells: those that arise from endogenous SSCs carrying the disease-causing mutation as well as those from the corrected SSCs introduced by transplantation. As a consequence, the semen of the father would contain a mixed population of spermatozoa and children conceived by natural conception could be derived from either a corrected or endogenous sperm cell. Although local irradiation has been demonstrated to be an effective measure to deplete the testis of germ cells in animal studies (Zhang et al., 2006; Herrid et al., 2009), it can have a deleterious influence on outgrowth of seminiferous tubules, especially in prepubertal testes (Jahnukainen et al., 2011). Besides, it may cause damages to surrounding organs and cells. Moreover, some endogenous spermatogonia might survive the irradiation and are still capable of developing into spermatids, thereby risking the transfer of the genomic aberrations. In this sense, the development of alternatives to exclude endogenous SSCs is needed to better strike the balance between the benefits of SSCT and the potential risks of the required total depletion of endogenous spermatogenesis. While SSCs have been demonstrated to be refractory to most non-viral transfection approaches (Kanatsu-Shinohara et al., 2005), novel electroporation devices that are currently being used in some laboratories may be the option to transfect SSCs with adequate efficiency (Zeng et al., 2012; Fanslow et al., 2014; Wu et al., 2015; Chapman et al., 2015; Sato et al., 2015). Alternatively, transfection of the mRNA instead of the corresponding DNA vectors has been shown to be more efficient for genome editing (Fanslow et al., 2014). Also the recently developed novel method regarding direct intracellular delivery of proteins might serve as another option for gene targeting (D’Astolfo et al., 2015). testicular sperm extraction (TESE) Can we grow sperm? A translational perspective on the current animal and human spermatogenesis models (2011) Initial germ cell to somatic cell ratio impacts the efficiency of SSC expansion in vitro (2017) Cryopreservation of human testicular diploid germ cell suspensions (2012) Slow cryopreservation is not superior to vitrification in human spermatozoa; an experimental controlled study (2015) What is the best cryopreservation protocol for human testicular tissue banking? (2013) Rapid screening of testicular tissue cryopreservation protocols https://onlinelibrary.wiley.com/doi/full/10.2164/jandrol.109.009324 Preserved seminiferous tubule integrity with spermatogonial survival and induction of Sertoli and Leydig cell maturation after long-term organotypic culture of prepubertal human testicular tissue https://academic.oup.com/humrep/article/32/1/32/2645566 Germ cell transplantation and neospermatogenesis https://link.springer.com/chapter/10.1007/978-3-319-42396-8_20 Transplanting germ cells including spermatogonial stem cells into the testis of an infertile recipient results in donor-derived spermatogenesis. Mammalian transgenesis by intracytoplasmic sperm injection (ICSI) http://science.sciencemag.org/content/284/5417/1180 exogenous DNA introduced with sperm head into oocyte 20% efficiency (20% of the offspring had integrated the transgene) Birth of mice after nuclear transfer by electrofusion using tail tip cells electrofusion instead of ICSI inject spermatids into a bacterial cell and inject oocytes too directed evolution of bacteria to facilitate transfection inside their cellular compartment? sperm-mediated gene transfer Intracytoplasmic sperm injection sperm-mediated gene transfer (ICSI-SMGT) The negative effects of exogenous DNA binding on porcine spermatozoa are caused by removal of seminal fluid https://kops.uni-konstanz.de/bitstream/handle/123456789/6707/Kang_2008_Theriogenology.pdf?sequence=1 Direct gene delivery to murine testis as a possible means of transfection of mature sperm and epithelial cells lining epididymal ducts https://onlinelibrary.wiley.com/doi/full/10.1111/j.1447-0578.2006.00117.x In vivo transfection of sperm cells has been developed as an alternative method for SMGT and can be carried out by direct gene delivery into an interstitial space in a testis (now called ‘testis‐mediated gene transfer [TMGT]’), into the vas deferens, or into seminiferous tubules. This review summarizes what has been achieved in the field of in vivo gene transfer using sperm cells. Huang et al. 31 used a unique method for gene delivery to germ cells within seminiferous tubules. First they infused seminiferous tubules of young 2-week-old mice with a transgene and then used in vivo EP to encourage transfection. Two weeks later the testes were harvested. ‘Transgenic testicular sperm’ expressing yellow fluorescent protein (YFP) were selected under observation using a confocal microscope and used for intracytoplasmic injection of sperm (ICSI). It is not known whether germ cells (spermatogonia) can be transfected using this seminiferous tubule-targeted gene transfer method. Almost all researchers suggest that it may be difficult to use, probably because Sertoli cells cover and shield stem cells from the inflow of the DNA present in the tubular lumen. This problem is easily solved if a plasmid construct carrying a spermatogoniaspecific promoter and reporter gene could be introduced into the seminiferous tubule with several enzymes, such as trypsin and collagenase, believed to digest cell–cell junctions and intercellular matrix, and subsequently the entire testis were in vivo electroporated. Gene transfer to epididymal spermatozoa by direct introduction of DNA into the interstitial space of mammalian testis DIRECT INTRODUCTION OF calcium–phosphate‐precipitated circular plasmid DNA into the interstitial space of a testis of an adult mouse was first carried out by Sato et al.13 The aim was to transfect testicular spermatozoa so that exogenous DNA could be transmitted to the offspring at fertilization. Polymerase chain reaction analysis revealed that the introduced DNA could be detected in epididymal spermatozoa freshly isolated from the caput and cauda epididymides as early as 6 h after injection and in ejaculated spermatozoa recovered from the uteri of females. These findings suggest that a DNA solution injected into a testis is transferred to the epididymal portion, where epididymal spermatozoa incorporate it. Ogawa et al.14 extended the study of Sato et al.13 and demonstrated that 80.0% (16/20) of F0 blastocysts derived from mating with males whose testes had been exposed to liposome/lacZ expression plasmid complex exhibited lacZ activity. These findings clearly suggest that mature epididymal spermatozoa incorporate DNA exogenously introduced into an interstitial space of a mouse testis, leading to the generation of transgenic blastocysts with relatively high efficiency. This technology has been termed ‘testis‐mediated gene transfer (TMGT)’ as an in vivo alternative to SMGT.14 Sato et al.15 showed that a single injection of circular plasmid DNA complexed with Lipofectin into mature mouse testes is sufficient for transfection of spermatozoa (epididymal spermatozoa), and for a relatively high efficiency (50–100%) of gene delivery to mid‐gestational fetuses (F0) obtained by mating injected males with normal females. However, the introduced DNA appeared to be present in a mosaic pattern in the TMGT‐derived fetal tissues because it was estimated to be present at less than one copy per diploid cell.15 Sato et al.16 also revealed that the exogenous DNA introduced into a testis was transmitted to at least the second generation. Furthermore, Sato et al.16 tested several commercially available reagents that are used for in vitro gene transfer to determine which one was the most effective in introducing high numbers of exogenous DNA copies into the fetal mouse genome using TMGT.19 Unfortunately they were unable to find any candidate reagents for this purpose. commercially available liposomes were tested DMRIE‐C and SuperFect Kojima et al.22 injected adenoviral DNA into mouse testes using the TMGT method and found that Leydig cells, but not Sertoli or spermatogonic cells, were efficiently infected. They claimed that gene transfer of adenovirus into a testis using TMGT might be effective for in vivo gene therapy for male infertility resulting from Leydig cell dysfunction. Retrovirus‐mediated gene delivery into male germ line stem cells A rapid and non-invasive selection of transgenic embryos before implantation using green fluorescent protein (GFP) Gene transfer to sperm and testis: Future prospects of gene therapy for male infertility (2008) Several researchers have reported testis-mediated gene transfer using the liposome method. As early as 3–4 days after microinjection of the exogenous gene mixed with cationic lipids into the seminiferous tubules, gene expression was observed within both immature and differentiated germ cells. By 40 days post-injection, the gene expression was restricted to the most immature germ cells in the basal portion of the seminiferous tubules [38]. Although transgene was transmitted to morula, blastocyst and mid-gestational fetuses, the ratio of animals carrying the exogenous gene decreased as they developed. Yonezawa et al. [55] reported that more than 80% of morula-stage embryos expressed the exogenous gene, but only some of the postpartum progeny were foreign-DNA-positive with a high incidence of mosaicism. Ogawa et al. [56] reported that repeated injection with linearized plasmid DNA containing the -galactosidase gene encapsulated with cationic liposome into testis of adult mouse via the scrotum resulted in transmission of the exogenous DNA sequences to blastocysts through fertilization. Overall, 80% of blastocysts derived from mating with males receiving testis-mediated gene transfer were positively stained with X-gal staining for -galactosidase gene expression. Sato et al. [57,58] also attempted to transfect testicular spermatozoa with plasmid DNA by direct injection into testes to obtain transgenic animals. When injected males were mated with superovulated females 2 and 3 days after injection, more than 50% gene transmission was achieved in the mid-gestational fetuses. The copy number of exogenous DNA in the fetuses was estimated to be less than 1 copy per diploid cell, and overt gene expression was not found in these fetuses. These findings suggested that plasmid DNA introduced into a testis is rapidly transported to the epididymis and then incorporated by epididymal spermatozoa; however, to generate transgenic offspring with stability, several modifications will be needed. lipofection + DNA + electroporation Stable gene expression in spermatogenic cells using electroporation would be facilitated by retroviral integrase gene co-transfection [66]. ICSI using spermatozoa with a transfected gene could produce transgenic mice [70]. [70] Huang Z, Tamura M, Sakurai T, Chuma S, Saito T, Nakatsuji N. In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially localized jellyfish fluorescent protein gene. FEBS Lett 487: 248-251 (2000). https://febs.onlinelibrary.wiley.com/doi/full/10.1016/S0014-5793%2800%2902271-7 Oatley et al. [72] evaluated the development of spermatogenesis in ectopically grafted neonatal bovine testicular tissue and investigated the utility of using electroporation to stably transfect spermatogonial stem cells within the graft with an exogenous gene. This technique could potentially be an alternative to testis-mediated gene transfer and pronuclear injection because of the high success rate of stable transgene chromosomal incorporation. The making of transgenic spermatozoa (2003) https://academic.oup.com/biolreprod/article/68/5/1477/2683352 Transgenesis via permanent integration of genes in repopulating spermatogonial cells in vivo (2008) https://www.nature.com/articles/nmeth.1225 electroporation of mouse testis genome integration, long-term siring of transgenic pups An efficient method for generating transgenic mice using NaOH-treated spermatozoa (2010) https://academic.oup.com/biolreprod/article/82/2/331/2557936 Efficient generation of transgenic mice by lentivirus-mediated modification of spermatozoa Lentiviral mediated transgenesis by in vivo manipulation of spermatogonial stem cells http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021975 A non-surgical approach for male germ cell mediated gene transmission through transgenesis https://www.nature.com/articles/srep03430 electroporation of testes Robust generation of transgenic mice by simple hypotonic solution mediated delivery of transgene in testicular germ cells https://www.sciencedirect.com/science/article/pii/S2329050117300359 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. overall efficiency to transmit a transgene in the first generation was found to be 46% An efficient method for generating a germ cell depleted animal model for studies related to spermatogonial stem cell transplantation Multispecies purification of testicular germ cells Robust generation of transgenic mice by hypotonic shock mediated transgene delivery in testicular germ cells cool stuff. does it work in humans? Bacterial magnetic particles improve testes-mediated transgene efficiency in mice Effects of mesoporous silica nanoparticles upon the function of mammalian sperm in vitro Improved exogenous DNA uptake in bovine spermatozoa and gene expression in embryos using membrane destabilizing agents in ICSI-SMGT Intratesticular injection followed by electroporation allows gene transfer in caprine spermatogenic cells (2018) https://www.nature.com/articles/s41598-018-21558-9 worked in goats Not all sperm are equal: functional mitochondria characterize a subpopulation of human sperm with better fertilization potential http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018112 are there other cells that have fertilization potential? non-germinal haploid cells? Measurement and potential applications of induced triploidy in Pacific salmon (1983) https://www.sciencedirect.com/science/article/abs/pii/0044848683900807 An efficient method for the generation of transgenic rats avoiding embryo manipulation https://www.sciencedirect.com/science/article/pii/S2162253117301208 testicular injection + electroporation Finite-element modelling and preliminary validation of microneedle-based electrodes for enhanced tissue electroporation http://ieeexplore.ieee.org/abstract/document/8037629/ Permeabilizing Soybean Protoplasts to Macromolecules Using Electroporation and Hypotonic Shock (1987) http://www.plantphysiol.org/content/83/1/24 Electroporation produced 10 times more uptake than hypotonic shock treatment. Use of stirred suspension bioreactors for male germ cell enrichment (2016) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495556/ lentiviral vectors injected into testis? Enhanced transgenesis by intracytoplasmic injection of envelope‐free lentivirus https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381737/ We demonstrate enhanced transgenesis in mice by intracytoplasmic injection of envelope-free lentivirus. Envelope-free lentivirus carrying the green fluorescent protein (GFP) gene under the control of the ubiquitin promoter (LVU-GFP) was microinjected into the cytoplasm of mouse zygotes prior to embryo transfer. Ninety-seven percent (31/32) of the adult mice were confirmed transgenic by PCR and Southern blot analysis; all founder mice express GFP when tail snips were examined by fluorescent microscopy prior to genomic DNA extraction. Production of transgenic nonhuman primates (2014) https://www.sciencedirect.com/science/article/pii/B9780124104907000141 looks like lentiviral vectors injected into oocytes during ICSI (zygote stage) has been the primary method Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells Lentiviral transgenesis in mice via a simple method of viral concentration http://www.theriojournal.com/article/S0093-691X(16)30131-5/abstract (unclear where they were injecting their lentivirus viruses) Based on 26 individual constructs and 627 live pups, we found that the overall transgenic rate was more than 30%, which is higher than obtained with pronuclear microinjection. In addition, we did not find any significant differences in transgenic efficiency when the size of inserts was less than 5000 bp. These results not only show that our modified method can successfully generate transgenic mice but also suggest that this approach could be generally applied to different constructs when the size of inserts is less than 5000 bp. Identifying genes important for spermatogonial stem cell self-renewal and survival (2006) Derivation of a human blastocyst after heterologous nuclear transfer to donated oocytes (2005) https://www.sciencedirect.com/science/article/pii/S1472648310609625 Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts https://www.researchgate.net/profile/Andrew_French/publication/5649628_Development_of_Human_Cloned_Blastocysts_Following_Somatic_Cell_Nuclear_Transfer_SCNT_with_Adult_Fibroblasts/links/09e4150f737cc76ed9000000.pdf Human oocytes reprogram somatic cells to a pluripotent state https://www.researchgate.net/profile/Scott_Noggle/publication/51699033_Human_oocytes_reprogram_somatic_cells_to_a_pluripotent_state/links/00b495253ff521d95b000000.pdf Embryo development after successful somatic cell nuclear transfer to in vitro matured human germinal vesicle oocytes Poor development of human nuclear transfer embryos using failed fertilized oocytes Viable embryos from injection of round spermatids into oocytes High-magnification ICSI overcomes paternal effect resistant to conventional ICSI Paternal effects acting during the first cell cycle of human preimplantation development after ICSI Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone Enhancement of embryo developmental potential by a single administration of GnRH agonist at the time of implantation Use of a modified intracytoplasmic sperm injection technique to overcome sperm-borne and oocyte-borne oocyte activation failures Improvement of delivery and live birth rates after ICSI in women aged> 40 years by ovarian co-stimulation with growth hormone Fertilizable oocytes reconstructed from patient's somatic cell nuclei and donor ooplasts Construction and fertilization of reconstituted human oocytes Reproductive semi‐cloning respecting biparental origin : A biologically unsound principle https://academic.oup.com/humrep/article/18/3/472/626024 In vitro development of non-enucleated rat oocytes following microinjection of a cumulus nucleus and chemical activation chimeras intended for human gamete production interspecies blastocyst complementation http://www.rbmojournal.com/article/S1472-6483(17)30260-2/fulltext The parthenogenetic development of rabbit oocytes after repetitive pulsatile electrical stimulation Sperm contributions to oocyte activation: more that meets the eye (2016) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785161/ Sperm-egg adhesion and fusion in mammals (2009) https://www.cambridge.org/core/journals/expert-reviews-in-molecular-medicine/article/spermegg-adhesion-and-fusion-in-mammals/4A30891A291E9F460F86D5E28CC171A3 The establishment of appropriate methods for egg-activation by human PLCZ1 RNA injection into human oocyte Transient exposure to calcium ionophore enables in vitro fertilization in sterile mouse models The combination of calcium ionophore A23187 and GM-CSF can safely salvage aged human unfertilized oocytes after ICSI Culture conditions affect Ca2+ release in artificially activated mouse and human oocytes Single Ca2+ transients vs oscillatory Ca2+ signaling for assisted oocyte activation: limitations and benefits Successful pregnancy after ICSI with strontium oocyte activation in low rates of fertilization