Neuron,

Vol. 4, 203-214,

February,

1990, Copyright

0 1990 by Cell Press

Lipofection of cDNAs in the Embryonic Vktebrate Central Nervous System Christine E. Holt, Nigel Carlick, and Department of Biology University of California San Diego La Jolla, California 92093

Elsa

Cornel

Summary Neurons from the embryonic brain of Xenopus were transfected in vivo with a vector expressing luciferase cDNA using a simple lipofection procedure. Luciferase activity was monitored quantitatively, and the protein was immunolocalized in whole-mount embryonic brains. Luciferase-expressing neurons were often intensely labeled, displaying a Colgi-like filling of their dendrites, axons, and growth cones. Luciferase expression could be targeted to the retina by simply removing the skin epidermis covering the area and exposing the whole embryo to the DNA-lipofectin mixture. Luciferase activity in transfected embryos rose to peak values during the first 48 hr posttransfection and was still detectable 28 days later. Cotransfection experiments in which embryonic nervous tissue was exposed simultaneously to two different genes, luciferase and chloramphenicol acetyltransferase, showed that transfected cells coexpressed the two genes at an extremely high frequency (85%100%). This offers the possibility of targeting functionally significant genes along with benign reporter genes in the developing CNS. Introduction An instructive ticular genes their translation

approach to determining what role parplay in developing systems is to express products abnormally during embryo-

genesis. In recent experiments of this nature, specific genes have been expressed ectopically in subsets of cells that would not normally express the gene (Feiler et al., 1988; Ruis i Altaba and Melton, 1989), grossly overexpressed in the cells that normally do express the gene product (Kintner, 1988), and repressed by anti-sense message (Cabrera et al., 1987; Ciebelhaus et al., 1988). The method commonly used for introducing foreign genes into cells in vivo is microinjection of DNA or RNA into the fertilized egg or early embryo. This strategy can lead to stable germline transformations (Palmiter and Brinster, 1986; Etkin and Pearlman, 1987) and to transient expression early in embryogenesis (Krieg and Melton, 1984). Transient expression of particular genes through RNA injections, for instance neural cell adhesion molecule (Kintner, 1988) and a homeobox gene (Ruis i Altaba and Melton, 1989), has been useful in analyzing the roles of these protein products in early events of embryogenesis. Levels of translation products from foreign transcripts rise soon after RNA injection into fertilized Xenopus eggs and

begin to fall around neurulation (Kintner, 1988);making microinjection the method of choice for studying early embryonic events. However, this short-lived expression is of limited use in studying later events of embryogenesis, such as the formation of nerve connections in the developing CNS, which begin in Xenopus at around 30 hr of development. Moreover, the introduction, by microinjection into the egg, of genes that lack tissue-specific promoters leads to widespread expression in many cell and tissue types throughout embryogenesis. Thus, any defects in the CNS may be secondary effects of earlier abnormal events. Ideally one would like to be able to introduce specific genes into particular parts of the neuroepithelium after neural induction has occurred so that one could probe such processes of neural development as commitment to cell fate and axonal pathfinding. Direct injections into single cells of the early nervous system is time-consuming, especially since the fraction of injected cells that actually express exogenous cDNA tends to be small Uonas et al., 1989). Retrovirusmediated transfer of DNA offers a promising approach to this end in birds and mammals and has yielded important information on lineage relationships in the CNS (Sanes et al., 1986; Price et al., 1987; Luskin et al., 1988). For the purposes of introducing different functional genes, however, there are problems with achieving high enough titers to infect a significant fraction of cells in vivo and engineering desired genes into the retrovirus without lowering its infection efficiency (Sanes, personal communication). Also, different cells vary in their susceptibility to retroviral infection, depending on whether they possess appropriate receptors, and not all types of cells can be subjected to viral infection. Some of these problems can be circumvented by transforming cells in culture and injecting them back into specific regions of the brain (Rosenberg et al., 1988). Standard transfection procedures for cells in culture, such as calcium phosphate precipitation, DEAEdextran, and electroporation, are not practical in vivo because of problems associated with toxicity and tissue accessibility. A mild and efficient DNA delivery reagent has recently been developed for transfecting a variety of different cell types in culture (Feigner et al., 1987; Chang and Brenner, 1989). This reagent, a cationic lipid comprising N-[I-(2,3-dioleoxyl)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dioleoxylphosphatidylethanolamine (DOPE; Lipofectin), forms positively charged liposomes that complex spontaneously with polynucleotides. These positively charged complexes interact with the negatively charged cell surface, fuse with the plasma membrane, and deliver functional DNA into the cell cytoplasm (Felgner and Ringold, 1989). In contrast to other transfection reagents, lipofectin is relatively nontoxic and, depending on the cell line, reportedly yields from 5- to IOO-fold

NWKJIl 204

Isolated Head

Figure ‘I. Schematic Representation of the Embryonic Manipulations Performed for DNA Transfection Experiments (A) Isolated brain preparations: The head of the embryo was cut off (broken lines indicate position of cuts) and (i) was transferred directly into transfection medium (DNA-Lipofectin) yielding an isolated head preparation with skin epidermis intact or (ii) the skin epidermis was removed to yield an isolated brain (including the eye primordia) preparation. (B) Targeted exposure through skin removal: The skin epidermis was removed (i) from the head region to expose the entire anterior neuroepithelium or (ii) from the eye primordia only. Whole embryos were then incubated in DNA-Lipofectin. (C) Direct injection: The DNA-Lipofectin mixture was injected directly into the iumen of the eye vesicle and spread into the lumen of the rest of the neural tube. The hatched area indicates the neuroepitheiium exposed to the injected transfection medium. (D) Eye grafting: Eye primordia with optic stalks attached were excised from donor embryos, incubated in DNA-Lipofectin for n8 hr, and returned to host embryos.

DNA/Lipofectin Stage 20-24 Embryo

isolated Brain

Skin from

Removed Head I

DNA/Lipofectin &

egraft Eye

I

I DNA/Lipofectin

higher transfection frequencies than either calcium phosphate or DEAE-dextran methods of DNA delivery. The present paper shows the introduction of genes into specific regions of the neuroepithelium via lipofection combined with embryonic surgery. The firefly (Photinus pyralis) cDNA encoding luciferase (de Wet et al., 1985, 1987) was transfected into cells of the presumptive eye and brain neuroepithelium by incubation with a luciferase-containing plasmid pRSVL(de Wet et al., 1987) and Lipofectin. Luciferase catalyzes a light-producing bioluminescent reaction that was used to quantitate the amount of enzyme present in the exposed neural tissue. The protein product was also visualized by immunocytochemistry. Our results show that a significant fraction of embryonic neurons become transfected after exposure to DNA and Lipofectin. Many newly differentiated and differentiating neurons express luciferase at high levels in their so-

mata and throughout their fine processes, revealing a Golgi-like filling of the whole ceil, including dendrites, axons, and growth cones. Moreover, neural tissue exposed to two plasmids encoding different reporter genes (luciferase and the bacterial gene chloramphenicol transferase [CATJ) contains many neurons that coexpress the two genes with extremely high efficiency, suggesting the potential value of this technique for assaying the behavior of developing neurons with misexpressed genes of interest. Results Luciferase Expression in Embryonic Brains When isolated brains were incubated with a plasmid containing luciferase cDNA (pRSVL) and Lipofectin (Figure IA), significant levels of luciferase activitywere found 48 hr later (Figure 2). No luciferase activity

Transfection

In Viva

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Luciferase

8ul s s .s 2 x t X"

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Brains

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Stage 20-24 isolated brains (see Figure IA, experiment ii) were incubated in transfection medium for 8 hr, transferred to 100% MR, and assayed at 40 hr posttransfection (equivalent to stage 40-41). Transfection media were as follows: DNA + LIPO, luciferase DNA (pRSVL; 15 up) and Lipofectin (45 up) in 500 ul of 100% MR; LIPO, Lipofectin only (45 pg in 500 ul of MR); DNA, pRSVL DNAonly (15 pg in 500 ul of MR); MR, 500 ~1 of 100% MR. Luciferase activity is expressed as the number of light units (LU) counted over 30 s. The luminometer was calibrated to yield 0.14 pg of pure luciferase protein per 1000 light units over 30 s. A total of 4 brains were homogenized per assay; 120 ~1 of the brain homogenate was used in each assay and contained 20 pg of soluble protein per assay. This standard set of conditions was used in the rest of the data presented here. The brains exposed to both DNA and LIP0 show luciferase activity in the range of 14 pg. Control brains (DNA, LIPO, and MR) yield background levels of activity. This type of experiment was repeated 6 times and consistently showed that the DNA + LIP0 brains became transfected, whereas the DNA controls did not. The total amount of luciferase activity varied from experiment to experiment: 416 yielded 1 x IO5 to 3 x 10s light units; l/6 had 5 x IO3 light units; 116 had 1 x IO6 light units (a range of 0.7 pg to 140 pg of luciferase per 20 ug of total protein).

could be detected when tissue was incubated with pRSVL alone. The total amount of luciferase activity varied widely from experiment to experiment and ranged from 0.7 to 140 pg of luciferase per 20 pg of total protein. This variability could be due to several factors, such as differences in the susceptibility of different batches of embryos to lipofection, nuclear uptake of the exogenous DNA, and differences in the replication efficiency of plasmid DNA (Marini et al., 1988). The data presented in Figure 2 are from an experiment run with a single batch of sibling embryos. This particular experiment was repeated several times, as were all of the experiments described in the subsequent sections (see Figures for number of times each experiment was repeated). Optimization of Transfection Efficiency To establish the conditions that give rise to optimal transfection frequencies, several experimental parameters were varied using the isolated head and brain preparations shown in Figure IA. Ratio of DNA to Lipofectin The ratio of nucleic acid to Lipofectin during transfec-

tion has been shown to be critical for optimizing transfection efficiency in cultured cells (Felgner et al., 1987). To determine the optimal ratio for embryonic Xenopus neural tissue, the amount of Lipofectin was kept constant (45 pg) while the amount of luciferase DNA was varied (Figure 3A). Maximal luciferase expression was found at a ratio of about I:3 (DNA:Lipofectin). This ratio was used in subsequent experiments. Luciferase activity decreased with levels of DNAabove 15 pg, suggesting that excess DNA inhibits transfection. These results are similar to those obtained in vitro (Feigner et al., 1987) and are consistent with the view that excess DNA reduces the net positive charge of DNA-Lipofectin complexes, thereby inhibiting their interaction with the negatively charged cell membrane (Felgner and Ringold, 1989). Enzyme Treatment Embryonic neuroepithelium possesses a layer of extracellular matrix components on its surfaces. We suspected that Lipofectin might interact more efficiently with cell membranes that had been “cleaned” by mild enzymatic treatment. Embryonic tissue was therefore treated for varying times in solutions containing trypsin in calcium-free solution (ATV solution), collagenase, ficin, and elastase before transfection. in general, enzymatic pretreatment led to a significant rise in luciferase activity; however, the amount of increase was variable. ATV pretreatment for 1.0-1.5 min, for example, increased activity 2- to IOO-fold over no enzymatic treatment (n = 8; Figure 3B). Collagenase, ficin, and elastase also produced increases in activity, but these were usually lower than those with ATV solution. In some cases, enzymatic pretreatment did not appear to elevate transfection rates (see Figure 3B, Fitin). Increasing the period of exposure to ficin and elastase (I mg/ml) from 5 to 30 min produced corresponding increases in luciferase activity from 1.5- to lcfold (data not shown). In contrast, increased ATV exposure (over 1.5 min) was harmful to the tissue and reduced luciferase activity. Time in Transfection Medium To determine the length of incubation in transfection medium yielding the highest levels of luciferase activity, embryonic heads were incubated in DNA-Lipofectin mixture for varying durations. Luciferase activity increased with incubations of up to 20.5 hr and fell off thereafter (Figure 3C). The fall-off may result from a reduction in viability or mitotic activity in isolated brains at stage 35/36. Therefore, it is unclear whether there is a maximal duration, but for at least 20 hr, transfection efficiency increased with time. We do not know the minimal exposure required to get significant luciferase expression, as we did not test exposures shorter than 8 hr. It is worth noting, however, that exposure durations of 2-4 hr of whole gastrula stage embryos show significant activity (Malone and Holt, unpublished data). Increasing DNA Concentration To test whether the total amount of DNA in the transfection medium limits transfection efficiency, the amount

NeLlrOn 206

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term expression. Therefore, long-term expression experiments were done using intact animals. Luciferase activity was monitored at varying intervals over a 14 day period on whole animals that had been transfected after the skin was removed from both eyes at stage 22 (Figure IB, experiment ii; Figure 4C). Activity rose during the first 4 days, remained at its peak value from 4 to 8 days, then dropped to just less than half the peak value by 14 days. In a second type of experiment to test for the stability of luciferase expression, the DNA-Lipofectin mixture was injected directly into the lumen of embryonic brains at stages 20-24 (see Figures IC and 4C). Luciferase activity rose

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Stability of Transfected DNA To examine the short-term pattern of luciferase expression, samples were assayed for activity during the 3 day period following transfection (Figure 4A). Significant levels of activity were detected at 8 hr (gl pg). Activity rose steadily thereafter to peak around 24-48 hr. At 56 hr, activity had usually declined Zo about half the maximal value. These experiments were performed using isolated heads, and we wondered whether the drop in activity after 56 hr might represent a decline in cell viability. Isolated heads do not survive beyond

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(A) The optimal DNA-Lipofectin ratio was determined by keeping the amount of Lipofectin constant (45 pg) while varying the amount of luciferase DNA. The highest levels of luciferase activity were obtained with ratios of around I:3 (DNA:Lipofectin). These and subsequent experiments in this figure were performed on isolated heads (see Figure IA, experiment i). Transfections were begun at stages 20-24, and luciferase assays were

performed 40-48 hr posttransfection. Each of the experiments presented was repeated 2-5 times. (B) Embryonic brainswere treated with different enzymes before DNA transfections were begun. Enzymatic treatments were as follows: None, no enzyme treatment; Trypsin, ATV solution (0.5 mg/ml trypsin, calcium-free) for 1.25 min; Collagenase, 1 mg/ml for 5 min; Ficin, 1 mg/ml for 2.5 min. Trypsin treatment consistently raised luciferase expression, whereas the other enzymes were more variable in their effects. (C) Embryonic brains were incubated in the DNA-Lipofectin (1:3) transfection medium for increasing durations. Activity rose with increasing incubation duration up to 15-22 hr and decreased thereafter. (D) The total amount of DNA was increased as was the amount of Lipofectin to maintain a fixed ratio; the total volume of the incubation medium remained the same (500 ~1). Peak values of activity were obtained with 50-90 pg of DNA. The experiment plotted here was run using the suboptimal I:2 ratio of DNALipofectin, which may partially account for the generally low levels of activity.

Transfection

In Viva

207

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Incubation of whole embryos in DNA-Lipofectin with the skin epidermis intact (intact) gave background levels of activity (compare with MR control). Incubations after complete skin removal from the head region (off head; see Figure 16, experiment i) resulted in high luciferaseactivity. Intermediate levels of activity resulted when the skin was removed from only the region covering the eyes (Off eyes; see Figure IB, experiment ii). The activity resulting in the latter experiment (Off eyes) probably reflects luciferase-expressing cells specifically located in the retina (see Figure 6 for immunolocalization).

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(A) Time course of luciferase expression over a 56 hr period following transfection using isolated embryonic brains (see Figure IA, experiment ii). Expression was detectable by 8.5 hr and rose steeply over the next 48 hr. Peak expression occurred between 24 and 56 hr. The decrease in activity seen after 248 hr, also detected in three other short-term experiments, may reflect a decrease in the viability of the isolated brain preparation rather than transiency of expression, since experiments with luciferase RNA injections into fertilized eggs suggest that the half-life of Iuciferase protein may be at least 4 days (data not shown). (B) Time course of luciferase activity in heads from intact embryos after the eyes were selectively transfected by localized removal of skin epidermis at stages 20-24. Expression rose over the first 2-4 days, reached a plateau between 4 and 8 days, and fell by 14 days. This pattern is similar to that seen with direct injections (see below). (C) Pattern of luciferase expression in brains from whole embryos after injection of pure DNA-Lipofectin into the lumen of the neural tube at stages 20-22. Luciferase activity rose over the first 2 days and reached peak levels between 2 and 8 days. Tranfected brains taken from live tadpoles at later times (IO, 16, and

Targeting Exogenous Genes to Specific Brain Regions We used two methods for targeting cloned luciferase DNA specifically to the eye region of the CNS. One way involved simply removing the skin epidermis covering the eye bud such that the only part of the neuroepithelium directly exposed to DNA-Lipofectin was the presumptive retina (see Figure IB, experiment ii). Samples treated in this way had levels of luciferase activity that were Jo-fold higher than those with the skin epithelium intact (Figure 5). lmmunocytochemistry revealed brightly labeled cell bodies located in the retina and optic axons coursing along the optic tract (see Figures 6 and 7). Retinal ganglion cell profiles are similar to those seen with direct intracellular injection of Lucifer yellow (Holt, 1989). Removal of the skin epithelium from the entire brain (Figure IB, experiment i) increased activity IO@fold over that with skin in place. These results indicate that the skin, which is composed of specialized mucous-secreting epithelial cells, is fairly refractory to transfection via lipofection. Attempts were not made to transfect other discrete

28 days) showed a maintained level of activity (0.28 pg of luciferase protein per brain), suggesting that this long-term expression may represent integrated DNA.

regions of the brain by localized skin removal; however, we envisage that such surgery could probably be used to target other areas such as the optic tectum, telencephalon, or hindbrain. Our second approach was to remove whole eye vesicles from embryos at stages 20-24, incubate them in the DNA-Lipofectin mixture for several hours, then return them to the embryos (Figure ID). This kind of procedure has been used in the past to administer other agents selectively to the embryonic eye (Holt, 1980; Holt and Harris, 1983). In these experiments, luciferase-positive cell bodies are confined to the retinas of the regrafted eyes and axons from retinal ganglion cells project along the optic tract to their midbrain target, the optic tectum (data not shown).

lmmunolocalization of Luciferase-Expressing Neurons The luciferase photoassay provides quantitative information on the total amount of luciferase activity present in tissue; however, it does not provide information about the number of cells transfected or the scale of expression in single cells. One would like to know, for example, whether many cells are producing low levels of luciferase or whether just a few cells are expressing very high levels? What is the rate of transfection (i.e., the ratio of positive to negative expressing cells)? To resolve these issues immunolocalization studies were performed. Transfected Cells Show Varying Levels of Luciferase Expression Luciferase immunocytochemistry of transfected neural tissues revealed manytransfected cells and a broad range in the intensity of staining (Figure 6). In some cells, the soma possessed a few punctate dots of label with little discernible label in the cell processes. This punctate labeling pattern is characteristic of luciferase expression in cultured mammalian cells in which the luciferase protein has been shown to be localized to peroxisomes (Keller et al., 1987). This was found to be the case here also, since double antibody labeling with anti-luciferase and an antibody to the peroxisome-targeting sequence showed colocalization of immunostaining (Figure 7). Cells showing this punctate type of labeling were characterized as low expressers and were found to constitute about 60% of the total population of transfected cells. Medium to high expressers displayed diffuse and often extremely bright labeling throughout the cell bodies and along the dendrites and axons. Fine structures such as growth cones were labeled in detail (Figure 6). Luciferase protein is probably too abundant to be sequestered into the limited number of peroxisomes in these medium to high expressers and therefore spills freely into the cytoplasm. Retinal ganglion cells in the eye frequently became transfected, and their axons could be seen coursing along the optic tract to their central target in the optic tectum (Figures 6 and 7). Other cell types in the retina, such as photoreceptors, interneurons, and Miiller cells, were also seen. Brightly la-

beled high expressers constituted about 40% of the total transfected population. Transfection Rate Isolated brain preparations (transfected without skin, ATV treatment, 15 pg of DNA: 45 vg of Lipofectin; Figure IA, experiment ii) had 80-100 luciferase-positive cells per half brain (on average). The total number of cells that constitute half an embryonic brain, estimated from counts of nuclei in stained, sectioned material, is in the range of 104 cells. The transfection rate was therefore estimated to be around 1%. Counts of transfected cells were not made in material that was treated with optimal levels of DNA; however, the observation that luciferase activity with 70 ug of DNA in the transfection medium is 4- to 5-fold higher than that with 15 p.g of DNA (see Figure 3D), which was used in the samples to make the above calculation, suggests that transfection rates of 4%-5% may be achieved. The possibility that these increased levels of luciferase reflect an increase in the amount of protein produced per cell rather than an increase in the number of cells transfected cannot be ruled out. Knowing the average number of cells transfected per brain (approximately200) and the total amount of luciferase activity (approximately 28 pg per brain) gives an estimated 0.14 pg of luciferase protein (1000 light units) produced per transfected cell. The transfection rate of brains directly injected with DNA-Lipofectin was not calculated; however, given that activity levels in these preparations were around IO-fold lower than those in isolated brains, a reasonable estimate might be in the range of 0.1%. Cotransfection of CAT and Luciferase To test whether single cells can become transfected with the two different genes and coexpress them, plasmids containing CAT (pRSVCAT) and luciferase (pRSVL) were mixed in equal proportions during the transfection procedure. lmmunocytochemical localization showed that 100% of the cells expressing medium to high levels of luciferase (as judged by the intensity and pattern of label; see previous section) also coexpressed CAT (Figures 7 and 8). Of the low luciferase expressers, 85% coexpressed both genes. In the remaining 15%, cells were seen to express one of the genes, with no detectable staining for the second one. Of these single expressers, 77% expressed luciferase only. This bias may simply reflect the fact that the sensitivity of detection was greater for luciferase than for CAT (de Wet et al., 1987). In general, there was an excellent correspondence between the intensity of immunostaining of the two genes when coexpressed in the same cell. Thus, cells showing intense luciferase staining usually exhibited a similar intensity of CAT staining. This suggests that the two genes are expressed at similar levels. Comparison of Different To determine the relative terminal repeat promoter

Promoters strengths of the RSV of the pRSVL plasmid

long and

Transfection 209

Figure

In Viva

6. lmmunolocalization

of Luciferase

Protein

in Whole-Mount

Brain

Preparations

‘e (Figure (A)-(D) were transfected as isolated brains (Figure IA, experiment ii) and in (E) transfection was targeted specifically to the stage 40. IB, experiment ii). (A, B, and C) Neurons expressing luciferase located in the mid-brain region of embryos at approximate (A) and (B) show clusters of brightly labeled somata with fine processes extending from them. In the area shown in (B) there i ! approxient cells. mately 14 labeled neurons (many of them are beyond the plane of focus). Note the range in the labeling intensity of difi (D) Luciferase-positive growth cones in the brain. Fine filopodia and lamellopodia are visible (arrows). (E) Luciferase-positive optic fibers (arrows) coursing through the optic tract (OT) and turning into the optic tectum (Tee). 01 ne of the axons is tipped with a growth cone (gc). These labeled axons originated from retinal ganglion ceils that were targeted for e, cpression by removing the skin epidermis from the eye region (Figure IB, experiment ii).

Figure

7. Coexpression

of Luciferase

and

CAT Proteins

in the

Retina

and

Localization

of Luciferase

to Peroxisomes

(A) and (6) show a single retinal ganglion cell (RGC) coexpressing luciferase (A) and CAT (B). The cell body, located in the retina, is brightly labeled and possesses a few dendrites; the axon (Ax) travels out of the retina and into the brain, where it terminates in a growth cone (not shown) in the chiasm region. (C) Punctate luciferase immunostaining in a single cell (low expresser). (D) Same cell as (C) immunostained with an antibody to the peroxisome-targeting signal. The pattern of labeled spots is very similar with the two stains (see arrows), showing that luciferase is localized in peroxisomes.

the SV40 early promoter, also linked to the luciferase cDNA (pSV2L), the two plasmids were transfected into embryonic brains in parallel. pRSVL-transfected brains yielded approximately 12 times more luciferase activity than pSV2L-transfected brains. This result indicates that in Xenopus CNS tissue, the RSV long terminal repeat promoter is significantly stronger than the SV40 promoter. Two other promoters, the Xenopus heat shock promoter (HSP70; Bienz, 1986) and the promoter for elongation factor-la (Krieg et al., 1989), were tested upstream of the b-galactosidase gene. Neither of these constructs yielded more than 1 or 2 fi-galactosidase-positive cells per transfected brain when in-

troduced into embryonic tissue via the lipofection procedure. However, the rates of transfection with the two different reporter genes are not directly comparable because the threshold for detectability of j3-galactosidase may be higher than that for luciferase. RNA Transfections To determine whether RNA can also be lipofected into embryonic neuroepithelial ceils, as has been demonstrated in culture (Malone et al., 1989), isolated brains were subjected to the transfection procedures described previously, but synthetic luciferase RNA was used instead of DNA. RNA-transfected brains showed

Transfection

In Vivo

211

Figure 8. Coexpression rons of the Midbrain

of Luciferase

and

CAT Proteins

in Neu-

(A) Luciferase expression seen with a rhodamine-selective filter. (6) CAT expression visualized with a fluoroscein-selective filter. The same field is shown in (A) and (B). Note the corresponding localization of label in the neurons and their processes.

appreciable levels of luciferase activity at 18 hr and 24 hr posttransfection (data not shown). Immunocytochemistry showed that RNA-transfected cells were all of the low expresser class (the luciferase was localized to peroxisomes), and no cells were found to exhibit the intense, diffuse staining seen with DNA indicative of high levels of expression. This suggests that functional RNA can be introduced into cells via lipofection but that the number of molecules transferred is not enough to transcribe levels of the protein sufficient to swamp the peroxisomal labeling. Discussion The present results show that foreign DNA can be introduced into cells in selected regions of the embryonic CNS using a lipofection procedure. Luciferase cDNA introduced in this manner is expressed at high levels in many neuronal cells and is distributed throughout the entire cytoplasm, enabling the visualization of

the fine processes of neurons with indirect immunofluorescence. Embryonic brains exposed to two different plasmids exhibit single neurons that coexpress the two markers at a high frequency. Enzymatic pretreatment of tissues usually increases the efficiency of transfection significantly. Thus, we think it is important that cells exposed to the DNALipofectin mixture have relatively clean plasma membranes. The resistance of the skin epithelium to lipofection may be related to the fact that it is composed of specialized mucous-secreting cells. The external mucous probably prevents liposomes from interacting and fusing with the plasma membranes. Luciferase activity in brains following direct injection of DNA-Lipofectin into the lumen was generally2 orders of magnitude less than that with isolated (enzymetreated) brain preparations. The reason for this lowered transfection efficiency might be extracellular materials along the ventricular surface of the neuroepithelium, and perhaps within the luminal fluid, exerting an inhibitory effect on the interaction of DNA-Lipofectin complexes with the cell surfaces. Indeed, the presence of charged serum proteins severely reduces DNA-Lipofectin interactions in cell culture (Felgner et al., 1987). This problem might be overcome by flushing the lumen with an enzymatic solution just prior to injection of DNA-Lipofectin. Luciferase contains a peroxisomal-targeting signal that localizes it to peroxisomes (Gould et al., 1987, 1989) in the firefly lattern organ and mammalian cells in culture (Keller et al., 1987; de Wet et al., 1987). Punctate labeling characteristic of peroxisomes was indeed seen in the low expressing labeled cells. The CAT proteins, however, was diffuse even in low expressing cells; this is indicative of its cytoplasmic localization and lack of any organelle-targeting sequence. The finding that a large fraction of the high expressing neurons transfected with the luciferase-containing plasmid expressed luciferase diffusely through their cytoplasm, extending into fine processes, probably reflects the fact that there is an overabundance of Iuciferase saturating the peroxisomal binding in these high expressing cells. The average amount of luciferase expressed per cell was estimated to be 0.14 pg or 1.5 x IO6 molecules. Cells expressing high levels of luciferase may in fact contain greater than twice this amount. The observation that most of the transfected cells coexpressed luciferase and CAT after simultaneous Iipofection with the two cDNA-containing plasmids is similar to results reported for mammalian cells in culture and gene cell, cence levels and the might

with other transfection techniques (Southern Berg, 1982; Gould et al., 1987). Furthermore, each was expressed to a similar extent in the same as judged by the intensity of immunofluoresstaining. All cells expressing medium to high of luciferase activity also expressed CAT activity, vice versa. Both genes were under the control of same promoter (RSV long terminal repeat); one therefore predict that competition for the same

NWKW 212

transcription factors would reduce the levels of expression. However, this did not seem to be a problem, since the fraction of cells expressing high levels of foreign proteins appeared to be the same in tissue transfected with a single plasmid and that transfected with two plasmids. The coexpression results can be exploited in the future by cotransfecting cells with a benign reporter gene, such as luciferase, along with a gene suspected of having particular functional significance. High levels of expression of luciferase activity would be indicative of correspondingly high levels of expression of the putative functional gene and could be used to monitor perturbations in cellular morphology that might result from gene misexpression (experiments of this sort are in progress). Luciferase activity rose steeply soon after transfection and reached peak levels by around 48 hr. Activity usually began to decrease by around 4-8 days, yet was still detectable after 4 weeks. This indicates that there is an early transient phase of luciferase expression followed by a long duration of stable expression and suggests that some of the plasmid DNA may become stably integrated into the genome of transfected neurons. However, additional experiments must be done to establish whether this is the case. Alternative explanations for this long-term expression are that the protein itself is very stable, that the plasmid DNA remains extrachromosomal and continues to be expressed, and that the injected DNA-Lipofectin mixture within the brain lumen continues to cause new transfection events. A goal of the present study was to develop a system whereby foreign genes could be targeted to discrete regions of the CNS. The observation that the skin effectively acts as a barrier blocking transfection of the underlying neuroepithelium led us to remove the skin covering the presumptive eye region, thereby selectively exposing the presumptive retina to DNALipofectin. This manipulation resulted in the localized expression of luciferase protein in cells of the retina. Moreover, the optic axons of high-expressing retinal ganglion cells could be traced in the brain, providing the potential for examining the processes involved in pathfinding and target selection through misexpression of developmental genes. We envisage that lipofection will be useful for targeting specific genes into other discrete areas of the developing CNS in Xenopus and in other vertebrate species. Experimental

Procedures

Preparation of Tissue Embryos were obtained

from hormone-stimulated matings of adult Xenopus frogs (frog colony maintained by N. Spitzer’s laboratory, UCSD). Embryos ranging from stage 20 to stage 24 (Nieuwkoop and Faber, 1956) were anesthetized in MS222 (1:10,000; Sandoz) in 100% MR (Gimlich and Cerhart, 1984) and pinned down in a Sylgard-coated dish. Four different types of embryonic preparation were used (see Figure 1). -Isolated heads and brains: These were prepared by severing the entire head from the embryo with a single transverse cut 50-100 Urn posterior of the eye vesicles and then removing the

cement gland and adhering endodermal tissue (Figure IA!. These isolated heads were sometimes bisected along the midbrain to yield two half brains with a single eye attached. The skin epithelium covering the head was either left intact (Figure IA, experiment i) or removed completely with fine minuetin pins (Figure IA, experiment ii). -intact embryos lacking skin on brains and eyes: The skin epithelium was peeled away from the entire anterior neuroepithelium or from only the two eye primordia in otherwise intact embryos (Figure IB). -Direct injections: Embryos received direct injections of DNALipofectin into the lumen of the neural tube (see section below). -Incubated and regrafted eyes: Eye primordia (without skin) were dissected free from embryos, incubated in transfection medium, and after several hours, washed in 100% IMR and replaced in host embryos using surgical procedures previouslydescribed (Holt and Harris, 1983). Isolated brain preparations and intact embryos were treated with one of several enzymes (collagenase [I mgiml], trypsin CO.5 mg/ml] in calcium-free solution [AlV solution; Irvine Scientific], ficin [Img/ml], elastase [I mgiml; Sigma], and trypsin with calcium [I mg/ml]), for varying times (I-30 min) and washed in 100% MR just prior to the start of transfection. All transfections were begun when the cells in the anterior neuroepithelium were still actively dividing (stages 20-24). Cells in the retina are first born at stages 25-26 (Holt et al., 1988), around the time when the first axon tracts begin to appear in the brain (unpublished data).

Transfection

Procedure

The DNA transfection procedure used was essentially a modified version of that described by Felgner et al. (1987) to transfect cultured cells. The plasmid DNAwas mixed with a synthetic cationic lipid preparation called Lipofectin (I:1 DOTMA: DOPE; Bethesda Research Laboratories) at a ratio of I:3 (this ratio was determined as giving optimal transfection rates; see Results). In a typical experiment, 15 bg of DNA was mixed with 250 ~1 of 100% MR, and 45 pg of Lipofectin (1 mg/ml) was added to 250 PI of 100% MR. These two solutions were mixed together in a 1 ml well 15-30 s before the embryonic tissue was added. Transfection plates were placed on a rotating table and incubated for 8-20 hr at room temperature. After transfection, embryos were transferred to fresh 100% MR and allowed to grow for an additional 20-30 hr. Transfected tissue was assayed for P. pyralis luciferase activity (de Wet et al., 1987) using a Monolight 2001 luminometer (Analytical Luminescence laboratories) with automatic injection of substrate and integration of counts over a 30 s interval. This luminometer was calibrated to yield 1000 light units per 0.26 pg of pure luciferase protein over a IO s integration (S. Subramani, personal communication). Our own readings, taken over a 30 s integration, yielded a conversion factor of 1.8x, giving 1000 light units per 0.14 pg of luciferase protein (1.5 x 106 molecules) per 30 s. The total amount of protein per sample was determined using a standard Bio-Rad protein assay.

Piasmid

DNA

Isolation

and Purification

Two plasmids were used to monitor transfection efficiency. pRSVL (de Wet et al., 1987) and pRSVCAT (gifts from S. Subramani) contain the luciferase and CAT genes, respectively, expressed under the control of the RSV long terminal repeat promoter. A IacZ-containing plasmid driven by elongation factor-la promoter (gift from P. Krieg) was found to be much less efficient. Plasmid DNA was purified from E. coli grown in superbroth. The plasmid DNA was banded two times in a cesium chloride-ethidium bromide gradient and was precipitated with ethanol prior to use in transfections. A plasmid containing the SV40 early promoter upstream of the luciferase cDNA (pSV2L; de Wet et al., 1987; gift from S. Gould) was used to compare activity with the RSV long terminal repeat promoter (pRSVL).

RNA Synthesis, A plasmid promoter

Purification,

and Transfection

expressing the luciferase (Malone et al., 1989; gift

cDNA from the phage T7 from I. Verma) was used to

Transfection 213

In Viva

transcribe luciferase RNA in vitro. The method of RNA synthesis and purification was similar to that described previously (Krieg and Melton, 1984; Malone et al., 1989). RNA transfection procedures were similar to those used with DNA. Direct Injection into Embryonic Brains To assay the stability of luciferase expression, approximately 100 nl of DNA-Lipofectin was injected directly into the lumen of the anterior neural tube at early eyebud stages (stage 20-24). Lipofectin was mixed with DNA at a ratio of 3:l (15 pg:5 vg) without dilution with MR. Fast green dye was added directly to the DNA-Lipofectin droplet, giving a dark green/blue color, in order to monitor visually the injections of transfection solution. The tip of the microinjection needle was inserted into the eye vesicle, and the dye could be observed spreading into the contralateral eye vesicle lumen and through the lumen of the presumptive spinal cord during injection. lmmunocytochemistry Transfected tissue was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7’4) at 4OC for 1 hr. Following fixation, the tissue was washed several times in serum-containing (10% fetal calf serum) buffer with 1% Triton X-100 at 33OC. Primary and secondary antibody incubations were performed for 1 hr each at 33OC in the presence of blocking serum and 1% Triton X-100. Brains without skin were reacted as whole mounts without further dissection; whole embryos and embryos with just their eyes exposed had their brains and eyes removed following fixation, before antibody staining. Samples were prepared as whole mounts and examined with fluorescence using appropriate filter sets. Luciferase was detected using a guinea pig anti-P. pyralis luciferase antibody (gift from Jon Singer) followed by a rhodamineconjugated goat anti-guinea pig Fab secondary antibody. CAT was detected using an anti-CAT monoclonal antibody (gift from S. Gould) followed by a fluorescein-conjugated goat anti-mouse Fab secondary antibody. Peroxisomes were localized using a peroxisome-targeting signal antibody (gift from S. Gould). Acknowledgments We would like to thank Bill Harris, Chris Kintner, Suresh Subramani, Charles Zuker, and Fred Gage for their valuable comments on the manuscript. We wish to thank Steve Gould, Brent Seaton, and Suresh Subramani for their gifts of cDNA constructs and antibodies and for valuable discussions and Clark Coffman, Bob Malone, and Phil Felgner for technical advice. We are also grateful to Jon Singer and Margie Adams for generously providing luciferase antibodies. This work was supported by the McKnight Foundation and the National Institutes of Health (#NS 23780-02). Received

August

23, 1989;

revised

November

28, 1989.

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