Microinjection Of Foreign DNA Research Articles

Manipulatingearly pig embryos

On the basis of established surgical procedures for embryo recovery and transfer, the early pig embryo can be subjected to various manipulations aimed at a longterm preservation of genetic material, the generation of identical multiplets, the early determination of sex or the alteration of the genetic make-up. Most of these procedures are still at an experimental stage and despite recent considerable progress are far from practical application. Normal piglets have been obtained after cryopreservation of pig blastocysts hatched in vitro, whereas all attempts to freeze embryos with intact zona pellucida have been unsuccessful. Pig embryos at the morula and blastocyst stage can be bisected microsurgically and the resulting demiembryos possess a high developmental potential in vitro, whereas their development in vivo is impaired. Pregnancy rates are similar (80%) but litter size is reduced compared with intact embryos and twinning rate is approximately 2%. Pig blastomeres isolated from embryos up to the 16-cell stage can be grown in culture and result in normal blastocysts. Normal piglets have been bom upon transfer of blastocysts derived from isolated eight-cell blastomeres, clearly underlining the totipotency of this developmental stage. Upon nuclear transfer the developmental capacity of reconstituted pig embryos is low and < 10% develop to morulae or blastocysts in vitro. Pig oocytes can be stimulated parthenogenetically and up to 10% grow to blastocysts in the in vitro culture. Sex determination can be achieved either by separation of X and Y chromosome bearing spermatozoa by flow cytometry or by analysing the expression of the HY antigen in pig embryos from the eight-cell to morula stage. Microinjection of foreign DNA has been successfully used to alter growth and development of transgenic pigs, and to produce foreign proteins in the mammary gland or in the bloodstream, indicating that pigs can be used as donors for valuable human pharmaceutical proteins. Another promising area of gene transfer is the increase of disease resistance in transgenic lines of pigs. Approximately 30% of pig spermatozoa bind considerable amounts of foreign DNA preferably at the post-acrosomal region, suggesting that transgenic animals can be obtained more efficiently than with the usual microinjection procedure. To increase gene transfer efficiency, considerable research efforts have been made to establish embryonic stem (ES) cells, but so far there is no definite proof of totipotency of the generated pig ESlike cells through viable chimaeras. In general, biotechnological procedures are much less advanced in pigs than in cows.

The future of transgenic farm animal production with emphasis on pigs.

Abstract Transgenic livestock already play an important role as models for human diseases, for producing recombinant pharmaceutical proteins, and in the future may serve as donors of organs for terminally ill patients and tissues and cells in regenerative medicine to cope with degenerative diseases and ultimately might contribute to satisfy the increasing demand for valuable animal derived proteins for an ever-increasing global human population. The first transgenic livestock were produced in 1985 by microinjection of foreign DNA into zygotic pronuclei. Since then, significant progress has been made with regard to methods that permit efficient and targeted insertion of precise modifications in the complex mammalian genomes. This in turn will further enhance the development of transgenic livestock with well-defined genetically engineered chromosomal loci that are not affected by neighbouring chromosomal sequences and without integration of undesired DNA sequences such as antibiotic resistant cassettes that hinder products of these animals to enter the market. Here, we review the recent advances of the methodologies for the production of transgenic farm animals which will be important in meeting global challenges in valuable animal protein production and will open new horizons for biomedicine.

A non-surgical approach for male germ cell mediated gene transmission through transgenesis.

Microinjection of foreign DNA in male pronucleus by in-vitro embryo manipulation is difficult but remains the method of choice for generating transgenic animals. Other procedures, including retroviral and embryonic stem cell mediated transgenesis are equally complicated and have limitations. Although our previously reported technique of testicular transgenesis circumvented several limitations, it involved many steps, including surgery and hemicastration, which carried risk of infection and impotency. We improved this technique further, into a two step non-surgical electroporation procedure, for making transgenic mice. In this approach, transgene was delivered inside both testes by injection and modified parameters of electroporation were used for in-vivo gene integration in germ cells. Using variety of constructs, germ cell integration of the gene and its transmission in progeny was confirmed by PCR, slot blot and immunohistochemical analysis. This improved technique is efficient, requires substantially less time and can be easily adopted by various biomedical researchers.

МЕТОДЫ ГЕНЕТИЧЕСКОЙ МОДИФИКАЦИИ И СЕЛЕКЦИЯ СЕЛЬСКОХОЗЯЙСТВЕННОЙ ПТИЦЫ (обзор)

The positive practical results in poultry breeding due to investigations on transgenesis and their prospects are discussed. The method for obtaining transgenic chickens and quail by microinjection of foreign DNA into eggs are described. It is based on a surgical operation, providing the access to the eggs to conduct microinjection of DNA and the natural formation of tertiary egg shells in the bird's genital tract. The microinjection into eggs at different stage of their development is considered in more detail. The transgenic chickens with humane growth hormone gene, b-galactosidase gene and humane b-interferon gene, and the transgenic quails with bovine growth hormone gene were produced by this technique. The quails with introduced bovine growth hormone gene and their offspring generations are characterized by an increased average egg mass and the higher immune status. Transgenesis used to transfer a target gene between two poultry lines, if compared with common breeding technologies, takes at least 7 to 8 years less and is probably less expensive, because there is no need in backcrosses to remove the unnecessary genes transmitted in the course of natural sexual hybridization. Nowadays, the transgenesis is very similar to directed, but indeterminate mutation. A transgene chances to insert genome, and that is why a lot of initial transgenic individuals are needed to achieve an inherited transgenesis.

The Yield and Composition of Milk from Transgenic Rabbits

Basic objective of this research was to compare the milk yield and composition of New Zealand White transgenic rabbit females expressing recombinant human factor VIII (hFVIII) in mammary gland during lactation with that of non-transgenic rabbit females of the same age during 30 days of lactation. Transgenic founders were generated by the microinjection of foreign DNA (mWAP-hFVIII gene construct) into the egg. F1, F2 and F3 generations of transgenic rabbits were obtained after mating of transgenic founder rabbits with non-transgenic rabbits. The amount of milk rejected was measured by weight-suckle-weight method at 10 th , 20 th and 30 th day of lactation. Quality of milk (content of fat, protein, lactose, dry ash, and some minerals) from transgenic and non-transgenic rabbit was also determined. Comparison of milk yield, determined by weight-suckle-weight method, showed significantly higher (p<0.05) milk production at day 20 of first lactation in non-transgenic females, but on the same day of second lactation higher milk yield was measured in transgenic ones. Significantly higher (p<0.05) content of milk fat and protein was determined in transgenic milk whilst higher content of lactose was found in non-transgenic milk. The content of minerals (calcium, phosphorus, magnesium and sodium) did not differ in both experimental and control groups. Our results showed that milk yield and composition of transgenic rabbit females (mammary specific transgenic over-expression of hFVIII) over several generations is only slightly and transiently different from milk yield of nontransgenic females, which had no significant consequence on the litter size and viability.

Transgenic farm animals: an update

The first transgenic livestock species were reported in 1985. Since then microinjection of foreign DNA into pronuclei of zygotes has been the method of choice. It is now being replaced by more efficient protocols based on somatic nuclear transfer that also permit targeted genetic modifications. Lentiviral vectors and small interfering ribonucleic acid (siRNA) technology are also becoming important tools for transgenesis. In 2006 the European Medicines Agency (EMEA) gave green light for the commercialistion of the first recombinant protein produced in the milk of transgenic animals. Recombinant antithrombin III will be launched as ATryn for prophylactic treatment of patients with congenital antithrombin deficiency. This important milestone will boost the research activities in farm animal transgenesis. Recent developments in transgenic techniques of farm animals are discussed in this review.

Health status of transgenic pigs expressing the human complement regulatory protein CD59

Microinjection of foreign DNA into pronuclei of zygotes has been the method of choice for the production of transgenic domestic animals. Following microinjection the transgene is randomly integrated into the host genome which can be associated with insertional mutagenesis and unwanted pathological side effects. Here, we evaluated the health status of pigs transgenic for the human regulator of complement activation (RCA) CD59 and conducted a complete pathomorphological examination on 19 RCA transgenic pigs at 1 to 32 months of age from nine transgenic lines. Nine wild-type animals served as controls. Expression levels of human complement regulator CD59 (hCD59) mRNA were measured by RT-PCR and distribution of hCD59 protein was determined by immunohistochemistry. Albeit variable transgene expression levels, no specific pathomorphologic phenotype associated with the presence of the transgene in all analyzed pig lines could be detected. Transgenic expression of this human RCA gene construct is not correlated with a specific pathological phenotype in pigs. This is crucial for the application of the technology and the use of transgenic pigs for biomedical and agricultural applications.

Application of transgenesis in livestock for agriculture and biomedicine

Microinjection of foreign DNA into pronuclei of a fertilized oocyte has predominantly been used for the generation of transgenic livestock. This technology works reliably, but is inefficient and results in random integration and variable expression patterns in the transgenic offspring. Nevertheless, remarkable achievements have been made with this technology. By targeting expression to the mammary gland, numerous heterologous recombinant human proteins have been produced in large amounts which could be purified from milk of transgenic goats, sheep, cattle and rabbit. Products such as human anti-thrombin III, α-anti-trypsin and tissue plasminogen activator are currently in advanced clinical trials and are expected to be on the market within the next few years. Transgenic pigs that express human complement regulating proteins have been tested in their ability to serve as donors in human organ transplantation (i.e. xenotransplantation). In vitro and in vivo data convincingly show that the hyperacute rejection response can be overcome in a clinically acceptable manner by successful employing this strategy. It is anticipated that transgenic pigs will be available as donors for functional xenografts within a few years. Similarly, pigs may serve as donors for a variety of xenogenic cells and tissues. The recent developments in nuclear transfer and its merger with the growing genomic data allow a targeted and regulatable transgenic production. Systems for efficient homologous recombination in somatic cells are being developed and the adaptation of sophisticated molecular tools, already explored in mice, for transgenic livestock production is underway. The availability of these technologies are essential to maintain “genetic security” and to ensure absence of unwanted side effects.

Comparison of in vitro development and gene expression of in vivo- and IVM/IVF-derived porcine embryos after microinjection of foreign DNA

We compared the developmental ability and gene expression of in vivo- and IVM/IVF-derived porcine embryos following microinjection with SV40-LacZ. A total of 412 IVM/IVF-derived and 129 in vivo-collected zygotes was used to examine developmental ability and gene expression following DNA microinjection. When either DNA injected or noninjected zygotes were cultured for 4 d in NCSU 23 followed by 5 d in Eagle's minimal essential medium (EMEM), the percentages of zygotes developing to blastocysts and hatched blastocysts were higher (P < 0.05) compared with groups cultured in NCSU 23 alone. The percentages of injected embryos reaching the morula and blastocyst stages were significantly lower (P < 0.05) than that of noninjected control embryos whether in vivo or IVM/IVF derived. The percentages of morula and blastocyst stage embryos expressing the gene were higher in the in vivo-derived embryos than in IVM/IVF-derived embryos. A lower proportion of (67 to 77%) mosaicism was observed in the in vivo-derived embryos than in IVM/IVF (90 to 100%) derived embryos. The total cell number of blastocysts cultured in both NCSU 23 and EMEM media was significantly higher than that of blastocysts cultured continuously in NCSU 23. Our results suggest that this dual culture system enhanced embryo viability following microinjection of foreign DNA.

Studies on the Production of Transgenic Mice

This paper describes attempts to identify parameters that are critical for succesful gene transfer into the germ line of mice by microinjection of foreign DNA into fertilized eggs. In the first stage of the study, 1470 zygotes were obtained and 1269 (86%) were microinjected with a human growth hormone gene construct. Of these zygotes, 769 (60%) proved to be alive after the injection, and 420 of them were transfered into the pseudopregnant recipients (54%). As a result, 70 offsprings were obtained, and out of these 3 (4%) proved to be transgenic. In the second stage of the study, 1246 zygotes were obtained and 1020 (81%) were microinjected with a human p53 gene construct. Of these zygotes 601 (58%) proved to be alive after the injection and 400 (66%) of them were transfered into pseudopregnant recipients. As a result, 105 (26%) offsprings were obtained, and out of these, 6 (5%) proved to be transgenic. Comparison of the ratios of transgenic mice in both groups showed no significant difference (P>0.05).

Specific Stages of Oocyte Maturity in Medaka to Obtain Efficient Foreign Gene Transfer by Microinjection.

Microinjection of foreign DNA into oocyte nucleus is a useful method for producing transgenic medaka. To find the most appropriate stage of oocyte maturity to obtain efficient foreign gene transfer, we microinjected the plasmid pHV-GH containing Drosophila Hsp70 promoter and rainbow trout growth hormone cDNA into medaka oocytes collected 20-6h before ovulation at intervals of 2 hours. Injected oocytes were cultured until ovulation, inseminated and incubated for 7 days. Oocytes collected 14-6h before ovulation matured in culture while most of those col-lected 20-16h before ovulation did not. The proportion of oocytes which were successfully fertilized and normally developed to the 7-day-old stage after microinjection were highest for oocytes collected at 8h before ovulation, as 79% were successfully fertilized and 58% developed normally. Embryos developed normally to the 7-day-old stage were assayed for the existence of foreign sequences by slot blotting. The proportion of embryos positive for foreign sequences among those assayed was highest (60%) for oocytes at 8 and 10h before ovulation. Thus, the gene transfer efficiency, ie. the ratio of positive embryos to injected oocytes, was highest (35%) at 8h before ovulation.

Developmental potential of early bovine zygotes submitted to centrifugation and microinjection following in vitro maturation of oocytes

This study was designed to investigate the potential use of in vitro matured, in vitro fertilized bovine zygotes for producing transgenic calves by microinjection of foreign DNA. In Experiment 1, the effect of centrifugation (4 min, 15,000 × g, 20°C) on in vitro derived bovine zygotes was evaluated. In vitro development from 2 to 8 cells was not affected (80 vs 78%) when control zygotes (n = 211) were compared with zygotes treated (n = 210) 18 h post insemination. In Experiment 2, the influence of the centrifugation alone on the developmental potential of embryos was evaluated in rabbit oviducts for 120 h. The percentage of control and treated zygotes that developed to 1, 2 to 8, 8 to 32 and more than 32 cells were 7, 54, 10 and 10% vs 7, 40, 11 and 10%, respectively. In Experiment 3, the effect of pronuclear injection with plasmid containing CRF (corticotropin releasing factor) gene or pOCAT 330 Δ1 plasmids; 2 μg/ml in Tris 10 mM, EDTA 0.2 mM, 18 to 20 h post insemination was evaluated by in vivo development in the rabbit oviduct. The embryos submitted only to centrifugation and vortexing resulted in a morula-blastocyst (> 32 cells) rate of 25% (n = 226) compared with the injected zygotes of which only 5% (n = 206) achieved the same stage. We conclude that in vitro produced bovine zygotes have a reduced developmental potential following microinjection, and this effect is not due to the centrifugation process.

Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.

Microinjection of foreign DNA into fertilized mammalian eggs is a convenient means of introducing genes into the germ line. Some of the more important parameters that influence successful integration of foreign DNA into mouse chromosomes are described. The effects of DNA concentration, size, and form (supercoiled vs. linear with a variety of different ends) are considered as well as the site of injection (male pronucleus, female pronucleus, or cytoplasm) and buffer composition. The optimal conditions for integration entail injection of a few hundred linear molecules into the male pronucleus of fertilized one-cell eggs. Under these conditions about 25% of the mice that develop inherit one or more copies of the microinjected DNA. The overall efficiency also depends on the choice of mouse strains; for example, generating transgenic mice that express foreign growth hormone genes is about eight times easier with C57/BL6 X SJL hybrid mice than with inbred C57/BL6 mice.