Transferring genes into cultured mammalian embryos by electroporation

Mammalian whole embryo culture (WEC) was developed long before transgenic and gene targeted animals are widely used. Electroporation (EP) into cultured rodent embryos has expanded the potential to analyze gene functions in mammalian embryos by transferring exogenous plasmid vectors or small nucleotides in region- and stage-specific ways. This method is quite simple, and therefore enables us to analyze gene functions more quickly than genetic manipulation. In this review, we introduce combinatorial methods of WEC and EP, and summarize various applications in developmental neurobiology.     

Differential expression and stability of foreign genes introduced into human fibroblasts by nuclear versus cytoplasmic microinjection.

We have compared cytoplasmic- and nuclear-delivered, glass needle-mediated microinjection protocols for their ability to support both transient and stable phenotypic expression of reporter gene constructs in non-immortalized human skin fibroblasts cultures. Microinjection of form I (covalently closed circular, supercoiled) plasmid pMC38 DNA into the nucleus of human cells resulted in high levels of transiently expressed p110gag-myc oncoprotein as detected by immunofluorescence microscopy. Likewise, the nuclear delivery of a plasmid construct bearing the entire simian virus 40 genome induced the formation of morphologically transformed foci in approximately 6% of the recipient cell population. In contrast, the introduction of plasmid DNA by the cytoplasmic route proved virtually incapable of supporting either transient gene expression or morphological transformation. In situ autoradiography of cells injected with 3H-labelled plasmid DNA revealed that whereas the material delivered directly into the nucleus was retained by this subcellular compartment for prolonged times (greater than or equal to 48 h), the radiolabelled DNA molecules introduced via the cytoplasmic route did not reach the nucleus and appeared to be substantially degraded within 8 h following injection. These results indicate unequivocally that nuclear injection is the route of choice when monitoring foreign gene expression in human cells.     

Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.     

Gain- and loss-of-function in chick embryos by electroporation

It remained very difficult to manipulate gene expression in chick embryos until the advent of in ovo electroporation which enabled the induction of both gain-of-function, and recently loss-of-function, of a gene of interest at a specific developmental stage. Gain-of-function by electroporation is so effective that it has become widely adopted in developmental studies in the chick. Recently, it became possible to induce loss-of-function by introducing an siRNA expression vector by electroporation. In this review, the methods of electroporation for gain-of-function and for loss-of-function by siRNA are discussed.     

Change in the radiation responses of oogonia in the embryos and fry of the fish Oryzias latipes.

Embryos of the fish Oryzias latipes were irradiated with 1000 R of X-rays 1 day before hatching,and the post-irradiation change in the female germ-cell population was observed. Scarcely any reduction in the number of oogonia was observed, but their proliferation was inhibited. Repopulation occurred between 12 and 20 days after hatching. These responses were quite different from those of germ cells in the irradiated fry (Hamaguchi and Egami 1975). Embryos and/or fry were also exposed to 1000 R of X-rays 1 day before hatching and 0, 1, 2, and 3 days after hatching. A comparison of their responses suggested that the change in the radiation responses of oogonia is correlated with the initiation of meiosis.     

Expression of SV40-lacZ gene in mouse preimplantation embryos after pronuclear microinjection.

In order to study the expression of an exogenous gene in developing mouse embryos during the preimplantation period, DNA carrying the SV40 early promoter fused with the Escherichia coli beta-galactosidase gene (lacZ) was microinjected into the pronucleus of fertilized mouse eggs. Expression of lacZ gene was detected by staining embryos with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as a substrate at pH 7.2. The embryos expressing the lacZ gene showed various intensities of blue staining, all showing a mosaic pattern. The exogenous gene was expressed from the 4-cell stage until the blastocyst stage. The proportion of embryos expressing the lacZ gene was maximal (38%) at the morula stage, and the expression was dependent on the presence of the SV40 promoter.     

Expression in cowpea seedlings of chimeric transgenes after electroporation into seed-derived embryos

Summary Cowpea (Vigna unguiculata Walp) embryos mechanically isolated from mature seeds and incubated in the presence of plasmid DNA harboring chimeric gus genes were shown to germinate into seedlings expressing -glucuronidase activity in a variety of tissues, including the apical meristem. Embryo electroporation in the presence of DNA and protectants such as spermine and Lipofectin^TM increased both the proportion of embryo-derived seedlings expressing the chimeric gene and the level of gene expression. Microscopic observations of thin sections showed that the blue crystals representing the end product of transgene activity on X-glu were exclusively located inside the treated cells. Histological localization of the blue dye crystals varied with the promoter used to drive the transgene.     

Localized electroporation: a method for targeting expression of genes in avian embryos

Avian embryos are a popular model for cell and developmental biologists. However, analysis of gene function in living embryos has been hampered by difficulties in targeting the expression of exogenous genes. We have developed a method for localized electroporation that overcomes some of the limitations of current techniques. We use a double-barreled suction electrode, backfilled with a solution containing a plasmid-encoding green fluorescent protein (GFP) and a neurophysiological stimulator to electroporate small populations of cells in living embryos. As many as 600 cells express GFP 24-48 h after electroporation. The number of cells that express GFP depends on the number of trains, the pulse frequency and the voltage. Surface epithelial cells and cells deep to the point of electroporation express GFP. No deformities result from electroporations, and neurons, neural crest, head mesenchyme, lens and otic placode cells have been transfected. This method overcomes some of the disadvantages of viral techniques, lipofection and in vivo electroporation. The method will be useful to biologists interested in tracing cell lineage or making genetic mosaic avian embryos.     

Expression of cloned immunoglobulin genes introduced into mouse L cells.

Functionally rearranged immunoglobulin heavy-chain (gamma 2b) and light-chain (lambda 1 and kappa) genes were introduced into mouse L tk- cells by co-transformation with the Herpes virus tk gene. Cloned cell lines were selected in HAT medium and tested for the presence of transfected immunoglobulin gene sequences by Southern blotting analysis. It was found that the gamma 2b gene was accurately transcribed at a low level in transfected mouse L cells and cytoplasmic gamma 2b, heavy-chain protein was detected by immunoprecipitation of cell extracts. Light-chain genes, on the other hand, were not accurately transcribed. Instead, lambda 1 or kappa RNA species were detected which were approximately 200 to 300 bases longer than the authentic mRNAs. These results suggest that the expression of rearranged heavy-chain and light-chain genes are controlled differently and that these differences can be seen in transfected, non-lymphoid cells.     

Expression of specific genes in early mouse embryos blocked by cytochalasin

Summary Mouse embryos at the two cell stage derived from C57BL/6 × C3H/Aa F₁-females heterozygous at the X-linked phosphoglycerate kinase locus (Pgk-1) were cultured continuously in the presence of cytochalasin B or D. Further cleavage of the two cell embryos was thus prevented and the embryos became polyploid during culture. The onset of expression of the maternally inherited Pgk-1 gene and of the paternally inherited glucosephosphate isomerase (Gpi-1) gene was determined in these polyploid embryos by cellulose acetate gel electrophoresis of single embryos. In contrast to euploid preimplantation embryos developing normally in utero or in culture without cytochalasins, expression of maternal Pgk-1 was never observed at days 4 and 5 of gestation in polyploid two cell embryos, showing that the Pgk-1 allele on the maternally inherited X chromosome is not activated independently of cytokinesis and morphogenesis. Expression of paternally derived Gpi-1, however, occurred in cleavage blocked embryos von day 5 of development. This may indicate that the activation of two genes which are both expressed during preimplantation development and which both code for glycolytic enzymes, is initiated by different signals.     

Introduction of DNA into chick embryos by in ovo electroporation

Gene transfer by in ovo electroporation has been applied to the study of developmental biology, especially to central nervous system (CNS) development. Plasmids are injected into the neural tube of stage 10 chick embryos, and a 25-V 25-msec square pulse is applied five times. Since DNA moves toward the anode, the cathode side of the neural tube is transfected, and the cathode side is used as the control. Expression of translation product of the introduced DNA is observed 2 h after electroporation, peaks around 20 h after electroporation and then weakens. Expression is transient when plasmids are used as expression vectors, but they are very suitable for studying early developmental events (e.g., gene expression cascades or interactions). Misexpression of Pax-5 is shown as an example.     

Expression of microinjected reporter gene lacZ during first cleavages of rabbit embryos

The growing use of reporter genes in a model transgenic system has been a fundamental approach of biology, but the strategy of transgenic embryo selection prior to transfer to foster mothers may greately increase the efficiency of transgenic livestock production. This study was conducted to assess the possibility of beta-galactosidase (beta-gal)-labeled transgenic rabbit embryo production. Rabbit zygotes were obtained from superovulated females after mating. Zygotes were microinjected into male pronuclei with pCMV-lacZ or SV40-lacZ constructs; while some embryos were co-injected with the scaffold attachment sequences--SAR. Embryos from control non-injected and microinjected groups were cultured in vitro. After 24, 48, 72, or 96 h of culture the embryos were stained with X-gal for beta-galactosidase. Transgenic embryos produced by pronuclear injection showed a discrete pattern of beta-galactosidase expression. The percentage of transgenesis with pCMV-lacZalone was 1.5, but with SAR sequences it increased to 4.2. In the case of SV40-lacZ construct, the efficiency of transgenesis was 2.3% and 4.1%, respectively. The mosaicism was 66.7% for all embryos injected with both constructs with or without SAR. The highest numbers of 100%-transgenic (non-mosaic) embryos were found in the group co-injected with SV40-lacZ and SAR. Transgenesis was seen as early as 24 h after injection, in four-cell embryos. Most of the microinjected embryos showed delayed development as compared with control. It was concluded that lacZ may serve as a reliable reporter for early transgenic embryo selection in order to produce transgenic animals.     

Linear DNA introduced into carrot protoplasts by electroporation undergoes ligation and recircularization

The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration. This paper provides evidence for the ligation of plasmid fragments by plant cells. Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24h later. Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression. Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40–50%. Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts. CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression. Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kbHind III fragment of pCaMVCATM. DNA isolated from nuclei of the protoplasts was used to transform competent cells ofEscherichia coli, and colonies were recovered that carried pGEM withHind III-CaMVCAT inserts. Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transformE. coli yielded an estimate of the frequency of plasmid ligation. A maximum of only 4% of the input linear DNA was recovered as circular molecules. This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms. Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning intoE. coli.