Improvement of Mouse Cloning from Any Type of Cell by Nuclear Injection

2018 ◽  
pp. 211-228 ◽  
Author(s):  
Sayaka Wakayama ◽  
Satoshi Kishigami ◽  
Teruhiko Wakayama
Keyword(s):  
Nuclear Injection

Expression and Stabilization of Microinjected Plasmids Containing the Herpes Simplex Virus Thymidine Kinase Gene and Polyoma Virus DNA in Mouse Cells

1983 ◽  
Vol 3 (4) ◽  
pp. 511-522
Author(s):  
Masaru Yamaizumi ◽  
Arthur L. Horwich ◽  
Frank H. Ruddle
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
T Antigen ◽  
Polyoma Virus ◽  
Large T Antigen ◽  
Early Region ◽  
Virus Origin ◽  
Mouse Cells ◽  
Simplex Virus ◽  
Nuclear Injection

To observe the effects of polyoma virus DNA on the expression of the herpes simplex virus (HSV) thymidine kinase (TK) gene early after transfer into TK-deficient mouse cells and the subsequent development of stable TK-positive transformants, we constructed a series of recombinant plasmids containing the herpes simplex virus TK gene joined with various segments of the polyoma virus genome and microinjected them into the nuclei or cytoplasm of LTK-A cells (TK − , APRT − ). The frequency of nucleus-injected cells expressing TK after 1 day, measured by autoradiography of cells incubated with [ 3 H]thymidine, increased approximately 30-fold when the plasmids contained the polyoma virus origin of replication. The origin includes sequences with homology to the simian virus 40 origin of replication and adjoining sequences, including a recently defined transcription-enhancing sequence. After microinjection of a single origin-containing plasmid molecule per cell, TK expression was detected in approximately 50% of the injected cells. When a larger number of origin-containing plasmid molecules were injected per cell, all cells showed early TK activity. When the entire polyoma virus early region was present, neighboring uninjected cells became TK positive. When plasmids were injected into the cell cytoplasm, approximately 400 times as many molecules per cell were needed to cause early TK activity. The frequency of stable transformation observed 2 weeks after nuclear injection of 10 to 20 polyoma virus origin-containing plasmid molecules per cell was at least 2 orders of magnitude greater than with plasmids containing the TK gene alone. The greatest enhancement of stable TK transformation was obtained with plasmids containing the origin alone, when the maximum frequency of stable transformation was 5%. The addition of the coding regions for the small and medium T antigens or the entire early region significantly decreased TK transformation frequency in a copy-dependent fashion. The timing of stabilization of TK-positive transformation was analyzed by releasing hypoxanthine-aminopterin-thymidine selection pressure at various times after microinjection, culturing the cells in nonselective medium, and assaying for TK activity. Stabilization was found to occur between 3 and 6 days after nuclear injection. Cells injected with a plasmid containing the origin and the early region were examined for expression of the large T antigen with polyoma virus antitumor serum and immunofluorescent staining. The expression of the large T antigen was clearly associated with a cytopathic effect. TK-positive clones observed 2 weeks after injection of the plasmid were uniformly T antigen negative. Cytotoxicity may be the result of plasmid replication and toxic levels of T antigen or TK. In addition, expression of the large T antigen may block stabilization by preventing the integration of origin-containing plasmid molecules.

scholarly journals Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA

Development ◽  
1988 ◽  
Vol 102 (2) ◽  
pp. 287-299 ◽  
Author(s):  
R.R. Franks ◽  
B.R. Hough-Evans ◽  
R.J. Britten ◽  
E.H. Davidson
Keyword(s):  
Dna Sequences ◽  
Larval Growth ◽  
Exogenous Dna ◽  
Zygote Nucleus ◽  
Cell Lineages ◽  
Larval Stages ◽  
Injection Process ◽  
End To End ◽  
Nuclear Injection ◽  
Cytoplasmic Injection

A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32% versus 8%) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36% of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12% of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmetamorphic juveniles was usually less than 0.1 (range 0.05-50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment.

scholarly journals Translocation of RNA-coated gold particles through the nuclear pores of oocytes.

1988 ◽  
Vol 106 (3) ◽  
pp. 575-584 ◽  
Author(s):  
S I Dworetzky ◽  
C M Feldherr
Keyword(s):  
Electron Microscopy ◽  
Xenopus Oocytes ◽  
Particle Distribution ◽  
Polyglutamic Acid ◽  
5S Rna ◽  
Nuclear Pores ◽  
Gold Particles ◽  
Specific Effects ◽  
Tracer Particles ◽  
Nuclear Injection

In the present study, various sized gold particles coated with tRNA, 5S RNA, or poly(A) were used to localize and characterize the pathways for RNA translocation to the cytoplasm. RNA-coated gold particles were microinjected into the nucleus of Xenopus oocytes. The cells were fixed after 15, 60 min, or 6 h, and the particle distribution was later observed by electron microscopy. Similar results were obtained with all classes of RNA used. After nuclear injection, particles ranging from 20-230 A in diameter were observed within central channels of the nuclear pores and in the cytoplasm immediately adjacent to the pores. Particles of this size would not be expected to diffuse through the pores, suggesting that some form of mediated transport occurred. In addition, it was found that the translocation process is saturable. At least 97% of the pores analyzed appeared to be involved in the translocation process. Gold coated with nonphysiological polynucleotides (poly[I] or poly[dA]) were also translocated. When nuclei were injected with either BSA-, ovalbumin-, polyglutamic acid-, or PVP-coated gold, the particles were essentially excluded from the pores. These results indicate that the accumulation of RNA-gold within the pores and adjacent cytoplasm was not due to non-specific effects. We conclude that the translocation sites for gold particles coated with different classes of RNA are located in the centers of the nuclear pores and that particles at least 230 A in diameter can cross the envelope. Tracer particles injected into the cytoplasm were observed within the nuclear pores in areas near the site of injection. However, only a small percentage of the particles actually entered the nucleus. It was also determined, by performing double injection experiments, that individual pores are bifunctional, that is, capable of transporting both proteins and RNA.

Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal

Development ◽  
1976 ◽  
Vol 36 (3) ◽  
pp. 523-540
Author(s):  
J. B. Gurdon
Keyword(s):  
Bovine Serum Albumin ◽  
Xenopus Laevis ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Oocyte Nucleus ◽  
Oocyte Cytoplasm ◽  
Nuclear Injection

A method is described by which nuclei associated with some cytoplasm can be rapidly prepared from a suspension of cells. The method involves the use of lysolecithin and bovine serum albumin. Oocytes of Xenopus laevis were injected with about 200 nuclei prepared from human He La cells by this method. Nuclei were deposited in oocyte cytoplasm, in the oocyte nucleus, or in the dispersed contents of a ruptured oocyte nucleus. Injected He La nuclei enlarge up to several hundred times in volume in the course of a few days. Their enlargement is associated with chromatin dispersion, increased binding of an acidic dye, and with the reduction in size, and eventual disappearance, of nucleoli. The amount of He La nucleus enlargement is much greater when the oocyte nucleus is ruptured. The fate of injected nuclei was followed by the use of HeLa nuclei whose DNA had been previously la belled with [3H] thymidine. La belled DNA does not pass from injected He La nuclei into the oocyte nucleus. Injected nuclei appear not to fuse with each other or with the oocyte nucleus. Nuclei prepared by the above method look morphologically healthy in oocytes cultured in vitro for up to one month after nuclear injection. Nuclei prepared by other methods, such as those involving the use of detergents, undergo deterioration within a few days after injection into oocytes.

31 FULL-TERM AND LIVE RABBIT CLONES PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 124 ◽  
Author(s):  
F. Du ◽  
J. Xu ◽  
S. Gao ◽  
L. Y. Sung ◽  
D. Stone ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Fusion ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Full Term ◽  
Fusion Method ◽  
Nuclear Injection

Transgenic/knockout (KO) rabbits can serve as an excellent animal model for human cardiovascular diseases (CVD) and other diseases. However, the production of transgenic/KO rabbits is hindered by low efficiency of traditional DNA microinjection and the unavailability of embryonic stem cell lines. An alternative approach is to produce transgenic/KO rabbits by somatic cell nuclear transfer (SCNT) using genetically modified somatic cells as nuclear donors. Our initial objective of the study was to prove the feasibility of cloning rabbits by SCNT because rabbit is a difficult species to be cloned. Rabbit oocytes were flushed from the oviducts of superovulated donors treated with the regime of follicle-stimulating hormone (FSH) and human choriani gonadotropin (hCG). Cumulus cells were then denuded from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in M199 + 10% fetal bovine serum (FBS) and confirmed by fluorescence microscopy. Cumulus cells used for nuclear donors were prepared from fresh cumulus-oocytes complexes. The donor nucleus was transferred into a recipient oocyte by either cell fusion or direct nuclear injection method. In the cell fusion method, a small donor cell with the diameter approximately 15–19 µm was transferred into the perivitelline space of an enucleated oocyte; subsequently the somatic cell-cytoplast pair was fused by applying three direct current pulses at 3.2 kV/cm for a duration of 20 µs/pulse. In the direct nuclear injection method, a mechanically lysed donor cell was injected into oocyte cytoplasm with the aid of a piezo-drill system. Fused embryos or injected oocytes were activated by the same electrical stimulation regime described above, and subsequently cultured in M199 + 10% FBS containing 2.0 mM 6-dimethylaminopurine (DMAP) and 5 µg/mL cycloheximide for 2 h. For the in vitro study, cloned embryos were cultured in B2 medium plus 2.5% FBS for 5 days (initiation of activation = day 0) at 38.5°C in 5% CO2 humidified air. For the in vivo study, cloned embryos were cultured for 20–22 h in vitro before transfer into pseudopregnant rabbit recipients. Pregnancy was monitored by palpation and/or ultrasound on Days 14–16 post embryo transfer (ET). The results (Table 1) show that the donor nuclei-introducing rate was higher with nuclear direct injection than with the cell fusion method (P < 0.05). There were no significant differences among subsequent cleavage and development to morula and blastocysts between both methods, although the development rates of cloned embryos via electrically mediated fusion were higher than those derived from the injection group. One recipient in the injection group (1/6, 17%) and six recipients in the fusion group (6/16, 38%) were diagnosed as pregnant. From the fusion group, one full-term but stillborn and one live and healthy clone rabbit were delivered on Days 33 and 31 post-ET, respectively. To our knowledge, this is the second report of full term development of cloned rabbit by somatic nuclear transfer cloning. Our further study is to clone live rabbit offspring with modified transgenic/KO somatic cell lines. Table 1. In vitro development of rabbit cloned embryos with cumulus cells as nuclear donors This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261–11.

Nuovi problemi derivanti dall’impiego delle tecniche di fecondazione artificiale

2006 ◽  
Vol 55 (4) ◽  
Author(s):  
A. Mancini ◽  
G. Grande ◽  
R. Festa ◽  
E.T.C. Giacchi ◽  
L. De Marinis ◽  
...  
Keyword(s):  
Reproductive Technologies ◽  
Round Spermatid ◽  
High Rate ◽  
Immunodeficiency Virus ◽  
Moral Problems ◽  
Obstetrical Outcome ◽  
Nuclear Injection ◽  
Round Spermatid Injection ◽  
Infertile Patients

Le tecniche di fecondazione artificiale (PMA) pongono al medico che opera nel campo delle metodiche della riproduzione e al bioeticista notevoli problemi di ordine morale, per la cui risoluzione è necessaria una base scientifica a monte della valutazione etica. Questo studio analizza, dunque, gli aspetti più propriamente scientifici in merito alla Intra Cytoplasmic Sperm Injection (ICSI). Tale tecnica è oggi quella più usata nei centri di PMA ed ha rivoluzionato l’approccio alla sterilità di coppia. Pur tuttavia essa pone notevoli problemi, sia in merito all’impiego di seme di soggetti infertili sia connessi alla stessa tecnica. Per quanto riguarda i primi, si distinguono aspetti correlati: 1. alla presenza di anomalie citogenetiche parentali e delle cellule spermatiche, onde è stato dimostrato che pazienti infertili ammessi a programmi ICSI hanno un alto tasso di aneuploidie negli spermatozoi, inversamente correlate con i parametri seminali e il numero di forme morfologicamente normali dopo selezione; 2. all’esistenza di microdelezioni del cromosoma Y. Su tale aspetto ci si sofferma, evidenziando il ruolo dei segmenti genici coinvolti, nonché il rischio di trasmissione della microdelezione ai figli concepiti via ICSI. Sono quindi descritte le principali problematiche connesse sia alla tecnica sia all’abilità dell’operatore, evidenziando il carattere di sperimentalità della tecnica in esame. Considerando, poi, i risultati delle procedure ICSI, occorre considerare sia il tasso di fecondazione e l’outcome ostetrico sia il seppur lieve aumento di incidenza di malformazioni ed anomalie citogenetiche. Inoltre, il tentativo di ottenere gravidanze da situazioni maschili sempre più severe, pone il problema di valutare i risultati in rapporto al quadro eziologico di partenza, ma vi sono poche osservazioni in tal senso. In conclusione vengono valutati i nuovi campi di indagine, con particolare riferimento alla round spermatid injection (ROSI), alla round spermatid nuclear injection (ROSNI) ed all’impiego di spermatogoni, fino alle forme di “semi-clonazione”, e gli ulteriori problemi che permangono ancora aperti, quali gli aspetti immunologici o la trasmissibilità dell’infezione da Human Immunodeficency Virus (HIV). ---------- The reproductive technologies (RTs) puts to the physician, involved in the reproductive medicine, and the bioethicist remarkable moral problems; therefore a solid scientific ground is necessary for an ethical evaluation. This study analyses the more properly scientific aspects, particularly related to Intracytoplasmic Sperm Injection (ICSI). Such technique, that is today the more used in the RTs centres, upsets the approach to couple sterility. It places, however, remarkable problems, both about the use of infertile man sperm and about the same technique. As to the first point, we can distinguish aspects connected to the presence of parental citogenetic anomalies and in sperm cells, so it was demonstrated that infertile patients admitted to ICSI programs have a high rate of aneuploidies in the spermatozoa, inversely correlated with the sperm parameters and the number of morphologically normal shapes after selection, and to the existence of micro-deletion of the Y chromosome. About this we meant to stop to us, evidencing the role of the deleted genic segments and the risk of transmission of the microdeletion to sons conceived via ICSI. Then the authors describe the major problems connected to the technique and to the ability of the operator, evidencing the experimentation of this technology. Considering, then, the outcomes of ICSI procedures, it is necessary to consider both the fecondation rate and the obstetrical outcome, and the slight increase of the incidence of malformations and citogenetical anomalies. Moreover, the attempt to obtain pregnancies from more and more severe male situations places the problem to estimate the outcome referred with the etiology, but there are only few observations about it. In conclusion we considered the new perspectives, specially about round spermatid injection (ROSI), round spermatid nuclear injection (ROSNI) and the use of spermatogonia, until “the semi-cloning reproduction” and the other problems still opened, as the immunological aspects or the transmission of the Human Immunodeficiency Virus (HIV).

scholarly journals Ooplasmic round spermatid nuclear injection procedures as an experimental treatment for nonobstructive azoospermia

1997 ◽  
Vol 14 (1) ◽  
pp. 55-62 ◽  
Author(s):  
Keiko Yamanaka ◽  
Nikolaos V. Sofikitis ◽  
Ikuo Miyagawa ◽  
Yasuhisa Yamamoto ◽  
Toshiko Toda ◽  
...  
Keyword(s):  
Experimental Treatment ◽  
Round Spermatid ◽  
Nonobstructive Azoospermia ◽  
Nuclear Injection

Reprogramming cattle somatic cells by isolated nuclear injection

1998 ◽  
Vol 10 (8) ◽  
pp. 645 ◽  
Author(s):  
Alan Trounson ◽  
Orly Lacham-Kaplan ◽  
Maria Diamente ◽  
Tiki Gougoulidis
Keyword(s):  
Somatic Cell ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cell Nuclei ◽  
Surface Appearance ◽  
Intracytoplasmic Injection ◽  
Fetal Fibroblasts ◽  
Passage Number ◽  
Nuclear Injection ◽  
Apparent Influence

The development of a somatic cell nuclear transfer procedure for the production of blastocyst stage cattle embryos is described. Bovine fetal fibroblasts were used for fusion experiments with surgically enucleated oocytes (cytoplasts) following the establishment of optimal parameters for electrofusion from isofusion contours. Fusion rates were increased by decreasing size of the cytoplasts used but cleavage was decreased by decreasing size of the cytoplast used (quarter, half and whole cytoplasts). The use of double cytoplasts did not improve cleavage, and development to blastocysts could not be achieved. In a comparison of electrofusion of fibroblasts with cytoplasts in the subzonal perivitelline space with intracytoplasmic injection of nuclei and parthenogenetically activated oocytes, 2%, 14% and 24% developed to blastocysts respectively. In the group injected with isolated nuclei, the passage number (4 to 9) had no apparent influence on developmental competence to blastocysts. The embryos produced by nuclear injection of somatic cell nuclei showed the normal pattern of cell surface appearance of TEC-3 and TEC-4 stage-specific epitopes during development, as seen in fertilized oocytes. We conclude that the nuclear injection of somatic cell nuclei is a relatively efficient way to clone bovine embryos.

Xenopus laevis transgenesis by sperm nuclear injection

2006 ◽  
Vol 1 (5) ◽  
pp. 2195-2203 ◽  
Author(s):  
Stuart J Smith ◽  
Lynne Fairclough ◽  
Branko V Latinkic ◽  
Duncan B Sparrow ◽  
Timothy J Mohun
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Injection
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