Structural data on the aging of diethylphosphoryl-butyrylcholinesterase.
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Butyrylcholinesterase↗
Biomedical subjects
O Lockridge
Publications and source records attributed to O Lockridge.
Acetylcholinesterase knockouts establish central cholinergic pathways and can use butyrylcholinesterase to hydrolyze acetylcholine.
Acetylcholinesterase is one of the most prominent constituents of central cholinergic pathways. It terminates the synaptic action of acetylcholine through hydrolysis and yields the choline moiety that is necessary for transmitter recycling. Despite these pivotal relationships, mice nullizygous for acetylcholinesterase established all principal anatomical components of central cholinergic pathways. No compensatory increase in the distribution of butyrylcholinesterase was detected. However, both the wild-type and nullizygous mice showed that butyrylcholinesterase enzyme activity extended to all parts of the brain receiving cholinergic innervation and that it could hydrolyze the acetylcholine surrogate acetylthiocholine. As opposed to acetylcholinesterase which was mostly of neuronal origin, butyrylcholinesterase appeared to be mostly of glial origin. These experiments lead to the unexpected conclusion that acetylcholinesterase is not necessary for the establishment of cholinergic pathways. They also show that butyrylcholinesterase can potentially substitute for acetylcholinesterase and that this enzyme is likely to play a constitutive (rather than just back-up) role in the hydrolysis of acetylcholine in the normal brain. The inhibition of butyrylcholinesterase may therefore provide a desirable feature of cholinergic therapies, including those aimed at treating Alzheimer's disease.
Acetylcholine↗
Effects of mutations of active site residues and amino acids interacting with the Omega loop on substrate activation of butyrylcholinesterase.
The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Omega loop. To determine whether the Omega loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10000-fold, indicating severe alteration in the binding area in W430A. The kcat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Omega loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.
Amino Acids↗
The active site of human paraoxonase (PON1).
Ideally we would like to treat people exposed to nerve agents with an enzyme that rapidly destroys nerve agents. The enzymes considered for such a role include human butyrylcholinesterase (BChE), acetylcholinesterase (AChE), carboxylesterase and paraoxonase (PON1). Success has been achieved in endowing BChE with the ability to hydrolyze organophosphates. The G117H mutant of BCHE hydrolyzes sarin and VX, whereas the double mutant G117H/E197Q hydrolyzes soman (Millard et al. Biochemistry 1995; 34: 15925-15933; 1998; 37: 237-247). However, the rates of organophosphate hydrolysis are slow and a faster organophosphate hydrolase is being sought. Native PON1 hydrolyzes paraoxon with a catalytic efficiency, of 2.4 x 10(6) M(-1) x min(-1), and our goal is to improve the organophosphate hydrolase activity of PON1. To achieve this we need to identify the amino acids in the active site of PON1. Using site-directed mutagenesis and expression in human 293T cells, we have identified the following eight amino acids as being essential to PON1 activity: W280, H114, H133, H154, H242, H284, E52 and D53. Fluorescence of PON1 complexed to terbium ion shows that at least one tryptophan is close to the calcium binding site.
Amino Acids↗
Evidence for nonacetylcholinesterase targets of organophosphorus nerve agent: supersensitivity of acetylcholinesterase knockout mouse to VX lethality.
The possibility that organophosphate toxicity is due to inhibition of targets other than acetylcholinesterase (AChE, EC 3.1.1.7) was examined in AChE knockout mice. Mice (34-55 days old) were grouped for this study, after it was determined that AChE, butyrylcholinesterase (BChE), and carboxylesterase activities had reached stable values by this age. Mice with 0, 50, or 100% AChE activity were treated subcutaneously with the nerve agent VX. The LD50 for VX was 10 to 12 microg/kg in AChE-/-, 17 microg/kg in AChE+/-, and 24 microg/kg in AChE+/+ mice. The same cholinergic signs of toxicity were present in AChE-/- mice as in wild-type mice, even though AChE-/- mice have no AChE whose inhibition could lead to cholinergic signs. Wild-type mice, but not AChE-/- mice, were protected by pretreatment with atropine. Tissues were extracted from VX-treated and untreated animals and tested for AChE, BChE, and acylpeptide hydrolase activity. VX treatment inhibited 50% of the AChE activity in brain and muscle of AChE+/+ and +/- mice, 50% of the BChE activity in all three AChE genotypes, but did not significantly inhibit acylpeptide hydrolase activity. It was concluded that the toxicity of VX must be attributed to inhibition of nonacetylcholinesterase targets in the AChE-/- mouse. Organophosphorus ester toxicity in wild-type mice is probably due to inhibition or binding to several proteins, only one of which is AChE.
Acetylcholinesterase↗
Predicted Michaelis-Menten complexes of cocaine-butyrylcholinesterase. Engineering effective butyrylcholinesterase mutants for cocaine detoxication.
Butyrylcholinesterase (BChE) is important in cocaine metabolism, but it hydrolyzes (-)-cocaine only one-two thousandth as fast as the unnatural (+)-stereoisomer. A starting point in engineering BChE mutants that rapidly clear cocaine from the bloodstream, for overdose treatment, is to elucidate structural factors underlying the stereochemical difference in catalysis. Here, we report two three-dimensional Michaelis-Menten complexes of BChE liganded with natural and unnatural cocaine molecules, respectively, that were derived from molecular modeling and supported by experimental studies. Such complexes revealed that the benzoic ester group of both cocaine stereoisomers must rotate toward the catalytic Ser(198) for hydrolysis. Rotation of (-)-cocaine appears to be hindered by interactions of its phenyl ring with Phe(329) and Trp(430). These interactions do not occur with (+)-cocaine. Because the rate of (-)-cocaine hydrolysis is predicted to be determined mainly by the re-orientation step, it should not be greatly influenced by pH. In fact, measured rates of this reaction were nearly constant over the pH range from 5.5 to 8.5, despite large rate changes in hydrolysis of (+)-cocaine. Our models can explain why BChE hydrolyzes (+)-cocaine faster than (-)-cocaine, and they suggest that mutations of certain residues in the catalytic site could greatly improve catalytic efficiency and the potential for detoxication.
Butyrylcholinesterase↗
Determination of the DNA sequences of acetylcholinesterase and butyrylcholinesterase from cat and demonstration of the existence of both in cat plasma.
Cat serum contains 0.5 mg/L of butyrylcholinesterase (BChE, EC 3.1.1. 8) and 0.3 mg/L of acetylcholinesterase (AChE, EC 3.1.1.7); this can be compared with 5 mg/mL and < 0.01 mg/L, respectively, in human serum. Cat BChE differed from human BChE in the steady-state turnover of butyrylthiocholine, having a 3-fold higher k(cat) and 2-fold higher K(m) and K(ss) values. Sequencing of the cat BCHE cDNA revealed 70 amino acid differences between cat and human BChE, three of which could account for these kinetic differences. These amino acids, which were located in the region of the active site, were Phe398Ile, Pro285Leu, and Ala277Leu (where the first amino acid was found in human and the second in cat). Sequencing genomic DNA for cat and human ACHE demonstrated that there were 33 amino acid differences between the cat and human AChE enzymes, but that there were no differences in the active site region. In addition, a polymorphism in intron 3 of the human ACHE gene was detected, as well as a silent polymorphism at Y116 of the cat ACHE gene.
Acetylcholinesterase↗
The butyrylcholinesterase K-variant shows similar cellular protein turnover and quaternary interaction to the wild-type enzyme.
A recent study has linked the butyrylcholinesterase (BChE) K-variant and the apolipoprotein epsilon4 isoform to late-onset Alzheimer's disease. These findings have been controversial and have led us to examine the differences between wild-type and K-variant BChE in enzyme activity, protein stability, and quaternary structure. J-variant BChE (E497V/A539T) was also studied because it is associated with the K-variant mutation. The K-variant mutation (A539T) is located in the C-terminal tetramerization domain. Wild-type, K-variant, and J-variant BChE were expressed in Chinese hamster ovary cells and purified. The purified enzymes had similar binding affinity (Km) values and catalytic rates for butyrylthiocholine and benzoylcholine. In pulse-chase studies the K-variant, J-variant, and wildtype BChE were degraded rapidly within the cell, with a half-time of approximately 1.5 h. Less than 5% of the intracellular BChE was exported. The C-terminal peptide containing the K-variant mutation interacted with itself as strongly as did the wild-type peptide in the yeast two-hybrid system. Both K-variant and wild-type BChE assembled into tetramers in the presence of poly-L-proline or the proline-rich attachment domain of the collagen tail. The native K-variant BChE in serum showed the same proportion of tetramers as the native serum wild-type BChE. We conclude that the K-variant BChE is similar to wild-type BChE in enzyme activity, protein turnover, and tetramer formation.
Adult↗
Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse.
We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/- mice had 50% of normal AChE activity and AChE -/- mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well.
Acetylcholinesterase↗
Postnatal developmental delay and supersensitivity to organophosphate in gene-targeted mice lacking acetylcholinesterase.
Acetylcholinesterase (AChE; EC 3.1.1.7) is the primary terminator of nerve impulse transmission at cholinergic synapses and is believed to play an important role in neural development. Targeted deletion of four exons of the ACHE gene reduced AChE activity by half in heterozygous mutant mice and totally eliminated AChE activity in nullizygous animals. Butyrylcholinesterase (EC 3.1.1.8) activity was normal in AChE -/- mice. Although nullizygous mice were born alive and lived up to 21 days, physical development was delayed. The neuromuscular junction of 12-day-old nullizygous animals appeared normal in structure. Nullizygous mice were highly sensitive to the toxic effects of the organophosphate diisopropylfluorophosphate and to the butyrylcholinesterase-specific inhibitor bambuterol. These findings indicate that butyrylcholinesterase and possibly other enzymes are capable of compensating for some functions of AChE and that the inhibition of targets other than AChE by organophosphorus agents results in death.
Acetylcholinesterase↗
Conserved aromatic residues of the C-terminus of human butyrylcholinesterase mediate the association of tetramers.
Human butyrylcholinesterase (BChE) in serum is composed predominantly of tetramers. The tetramerization domain of each subunit is contained within 40 C-terminal residues. To identify key residues within this domain participating in tetramer stabilization, the interaction between C-terminal 46 residue peptides was quantitated in the yeast two-hybrid system. The wild-type peptide interacted strongly with another wild-type peptide in the yeast two-hybrid system. The C571A mutant peptides interacted to a similar degree as the wild-type. However, the mutant in which seven conserved aromatic residues (Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564) and C571 were altered to alanines showed only 12% of the interaction seen with the wild-type peptide. The seven mutations (aromatics-off) were incorporated into the complete BChE molecule, with or without the C571A mutation, and expressed in 293T and CHO-K1 cells. Expression of wild-type BChE in these cell lines yielded 10% tetramers. The aromatics-off mutant formed dimers and monomers but no tetramers. The aromatics-off/C571A mutant yielded only monomers. Addition of poly-L-proline to culture medium, or coexpression with the N-terminus of COLQ including the proline-rich attachment domain (Q(N)PRAD), increased the amount of tetrameric wild-type BChE from 10 to 70%, but had no effect on the G534stop (lacking 41 C-terminal residues) and the aromatics-off mutants. Recombinant BChE produced by coexpression with Q(N)PRAD was purified by column chromatography. The purified tetramers contained the FLAG-tagged Q(N)PRAD peptide. These observations suggest that the stabilization of BChE tetramers is mediated through the interaction of the seven conserved aromatic residues and that poly-L-proline and PRAD act through these aromatic residues to induce tetramerization.
Amino Acid Sequence↗
Interaction between the peripheral site residues of human butyrylcholinesterase, D70 and Y332, in binding and hydrolysis of substrates.
Human butyrylcholinesterase displays substrate activation with positively charged butyrylthiocholine (BTC) as the substrate. Peripheral anionic site (PAS) residues D70 and Y332 appear to be involved in the initial binding of charged substrates and in activation control. To determine the contribution of PAS residues to binding and hydrolysis of quaternary substrates and activation control, the single mutants D70G/Y and Y332F/A/D and the double mutants Y332A/D70G and Y332D/D70Y were studied. Steady-state hydrolysis of the charged substrates, BTC and succinyldithiocholine, and the neutral ester o-nitrophenyl butyrate was measured. In addition, inhibition of wild-type and mutant enzymes by tetramethylammonium was investigated, at low concentrations of BTC. Single and double mutants of D70 and Y332 showed little or no substrate activation, suggesting that both residues were important for activation control. The effects of double mutations on D70 and Y332 were complex. Double-mutant cycle analysis provided evidence for interaction between these residues. The category of interaction (either synergistic, additive, partially additive or antagonistic) was found to depend on the nature of the substrate and on measured binding or kinetic parameters. This complexity reflects both the cross-talk between residues involved in the sequential formation of productive Michaelian complexes and the effect of peripheral site residues on catalysis. It is concluded that double mutations on the PAS induce a conformational change in the active site gorge of butyrylcholinesterase that can alter both substrate binding and enzyme acylation.
Binding Sites↗
Structural and hydration changes in the active site gorge of phosporhylated butyrylcholinesterase accompanying the aging process.
Wild-type (wt) butyrylcholinesterase (BuChE) and the E197D and D70G mutants were inhibited by diisopropylfluorophosphate (DFP) or soman under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process of DFP-phosphorylated enzymes (diisopropylphosphoryl-BuChE (DIP-BuChE)) was investigated. Hydrostatic pressure strongly increased the rate of aging of wt enzyme. The activation volumes (deltaV*) for the dealkylation reaction was -150 ml/mol for DIP-wtBuChE. On the other hand, pressure had little effect on the aging of the DIP-E197D mutant and no effect on the DIP-D70G mutant, indicating that the transition state of the aging reaction (dealkylation of an isoproxy chain) was associated with an extended conformation/hydration change in wtBuChE but not in mutants. The rate of aging decreased with osmotic pressure, supporting the idea that water is important for stabilizing the transition state. Molecular dynamics simulations were performed on the wtDIP adduct to relate the kinetic data to hydration changes in the enzyme active site gorge. The pH dependence of the melting temperature (Tm) of native and soman-wtBuChE, as determined by differential scanning calorimetry (DSC), indicated that the stabilization energy of aged BuChE is mainly due to the salt bridge between protonated H438 and PO-, with pK(H438) = 8.3. Electrophoresis under high pressure up to 2.5 kbar showed that aged wtBuChE did not undergo pressure-induced molten globule transition unlike the native enzyme. This transition was not seen for the mutant enzymes, indicating that mutants are resistant to the penetration of water into their structure. Our results support the conclusion that D70 and E197 are major residues for the water/H-bond network dynamics in the active site gorge of BuChE, both residues acting like valves. In mutant enzymes, mutated residues function like check valves: forced penetration of water in the gorge is difficult, release of water is facilitated.
Aging↗
Association of tetramers of human butyrylcholinesterase is mediated by conserved aromatic residues of the carboxy terminus.
Human butyrylcholinesterase (BChE) is composed predominantly of tetramers. Our laboratory has shown that up to 40 carboxy terminal residues of each subunit contribute to the stabilization of tetramers (R.M. Blong, E. Bedows, O. Lockridge, The tetramerization domain of butyrylcholinesterase is at the carboxy-terminus, Biochem. J. 327 (1997) 747-757). To better define the residues which participate in tetramer stabilization, the in vivo interaction of the BChE C-terminus 46 residue peptide was quantitated for wild type and mutant BChE using the yeast two-hybrid system. The wild type C-terminal peptides interacted with one another in this system. The K-variant (A539T) and C571A peptides showed interaction similar to that of the wild type. However, only 11.7% of the interaction seen with the wild type peptide was observed with the mutant in which seven conserved aromatic residues (Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564) had been altered to alanines (aromatics off mutant). When these seven mutations were incorporated into the complete BChE molecule and expressed in 293T cells, only monomers and dimers were observed. The addition of poly-L-proline to the medium of 293T cells expressing wild type BChE resulted in the increase of the tetrameric form, similar to that observed by Bon et al. (S. Bon, F. Coussen, J. Massoulié, Quaternary associations of acetylcholinesterase II. The polyproline attachment domain of the collagen tail, J. Biol. Chem. 272 (1997) 3016-3021) for acetylcholinesterase expressed in COS cells. However, no increase in tetramers was observed with poly-L-proline addition to the medium of 293T cells expressing the aromatics off BChE mutant. These observations suggest that the stabilization of BChE tetramers is mediated through the interaction of the seven conserved aromatic residues, Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564, and that the poly-L-proline induced increase in tetrameric BChE is mediated through these seven aromatic residues.
Amino Acid Sequence↗
Differences in active-site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase.
We examined the role of A328(F330) in the binding of various inhibitors to cholinesterases (ChEs) using human butyrylcholinesterase (BChE) mutants to determine if the conclusions drawn from studies with acetylcholinesterase (AChE) mutants could be extended to BChE. For huperzine A and edrophonium, the results obtained with AChE mutants could be directly correlated with those obtained with native ChEs and site-specific mutants of human BChE. Inhibition studies of ethopropazine with BChE mutants, where A328 was modified to either F or Y, suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active-site gorge showed that the poor inhibitory activity of ethopropazine towards AChE was due to the smaller dimension of the active-site gorge. The volume of the BChE active-site gorge is approximately 200 A3 larger than that of the AChE gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations. These results suggest that, although the overall scaffolding of the two enzymes may be highly similar, the dimensions and the micro-environment of the gorge play a significant role in determining the selectivity of substrate and inhibitors for ChEs.
Acetylcholinesterase↗
Human serum paraoxonase (PON1): identification of essential amino acid residues by group-selective labelling and site-directed mutagenesis.
Human serum paraoxonase/arylesterase (PON1, EC 3.1.8.1.) is a calcium-dependent enzyme which hydrolyzes a wide variety of organophosphates, including paraoxon, DFP, sarin and soman. Although the 3-D structure of PON has not yet been determined and its sequence shows no similarity with any other crystallized proteins, we undertook to identify some of its essential amino acid residues by two complementary approaches: group-specific labelling and site-directed mutagenesis. Group-specific labelling studies, performed on the purified native enzyme, indicated that one or more Trp, His and Asp/Glu are potentially important residues for PON activity. Based on these results, we identified some of these residues, conserved in the sequenced mammalian PON1, by site-directed mutagenesis. PON1 mutants were transiently expressed in 293T cells. The catalytic constants k(cat) and Km (relative to k(cat) and Km of the wild-type) determined with four different substrates (phenylacetate, paraoxon, diazoxon, chlorpyrifos oxon), were not significantly changed for the following mutants: W193A, W201A, W253A, H160N, H245N, H250N, H347N, E32A, E48A, D88A, D107A, D121A, D273A. By contrast, k(cat) was less than 1% for eight mutants: W280A, H114N, H133N, H154N, H242N, H284N, E52A and D53A. The essential amino acid residues identified in this work could be part of the PON1 active site, acting either as calcium ligands (E52 and D53?) or as substrate binding (W280?) or nucleophilic (His residues?) sites. However, we cannot rule out that the effects of mutations on catalytic properties resulted from a remote conformational change and/or misfolding of mutant proteins.
Amino Acids, Essential↗
Tryptophan residue(s) as major components of the human serum paraoxonase active site.
Serum paraoxonase (PON1, EC 3.1.8.1.) is a high density lipid- (HDL)-associated, calcium-dependent enzyme whose 3D structure, active site residues and physiological substrates are not known. The kinetic parameters k(cat) and Km (relative to k(cat) and Km of the wild-type), determined with four substrates (phenylacetate, paraoxon, diazoxon and chlorpyrifosoxon) were less than 1, and more than 100% for the W280A and W280F mutant enzymes, respectively. These results indicated that the aromatic/hydrophobic character of the amino acid in position 280 is essential for PON1 activity. In this study, we investigated whether this aromatic residue is in the PON1 active site. Group-specific labelling studies with N-bromosuccinimide, an oxidative agent of tryptophan, strongly suggested that one or several Trp could be in the active site of PON1 but we could not conclude either on the specificity of the labelling reaction or on the number of oxidized Trp. However, although PON activity was not altered by the hydrophilic tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl chloride (NBC), it was significantly reduced by the p-nitrophenylacetate analog 2-acetoxy-5-nitrobenzyl chloride (ANBC), whose hydrolysis by PON1 generated NBC in the active site. Moreover, since at least one calcium ion is present in the PON catalytic site, we attempted to probe the metal local environment using the calcium analog terbium. The luminescence spectrum of the PON terbium complex exhibited an emission peak at 545 nm characteristic of an aromatic residue (Trp and/or Tyr)-terbium interaction. In conclusion, both the results obtained with the mechanism-based inhibitor of PON1 (ANBC) and the calcium-binding site luminescent probe terbium support the hypothesis of the presence of at least one Trp residue in the PON1 active site. Trp residue(s) may be involved in the binding of aromatic substrates.
Acetates↗
Knockout of one acetylcholinesterase allele in the mouse.
One allele of the AChE gene (ACHE) was knocked out in embryonic stem (ES) cells by homologous recombination. The targeting vector contained 2 kb of a TK gene cassette for negative selection, 884 bp of ACHE including exon 1, 1.6 kb of a Neo(r) gene cassette for positive selection, 5.2 kb of the ACHE Bam HI fragment including exon 6, and 3 kb of Bluescript. The use of this vector deleted exons 2-5, which removed 93% of the ACHE coding sequence including the signal peptide, the active site serine, and the histidine and glutamic acid of the catalytic triad. The gene targeting vector was transfected into ES cells by electroporation. Colonies resistant to G418 and gancyclovir were screened for homologous recombination by Southern blotting. Out of 200 colonies, four were found to have undergone homologous recombination. These four ACHE (+/-) ES cell lines were expanded to provide cells for microinjection into C57Bl/6 mouse blastocysts. The injected blastocysts were implanted into pseudopregnant CD/l white mice. More than 200 injected blastocysts were transferred into 20 mice. More than 65 mice were born, of which 11 were chimeras. Chimeras were identified by their black and agouti coat color. Littermates were all black. Thus far, seven male chimeras have been bred with more than 130 C57Bl/6 females to generate 26 agouti mice out of 199 living offspring. This demonstrated that the ACHE (+/-) ES cells contributed to the germline. Offspring with agouti coat color have a 50% chance of carrying the knockout allele. The 26 agouti offspring were screened for an ACHE (+/-) genotype by tail biopsy PCR. Ten out of 26 agouti mice are heterozygous ACHE knockout mice, and they are healthy and alive at 29 days of age. We expect a phenotype to appear in nullizygous animals.
Acetylcholinesterase↗